U.S. patent application number 13/440940 was filed with the patent office on 2013-05-09 for method for the generation of compact tale-nucleases and uses thereof.
This patent application is currently assigned to Cellectis S.A.. The applicant listed for this patent is Claudia Bertonati, PHILIPPE DUCHATEAU, Jean-Charles Epinat, Alexandre Juillerat, George H. Silva, Julien Valton. Invention is credited to Claudia Bertonati, PHILIPPE DUCHATEAU, Jean-Charles Epinat, Alexandre Juillerat, George H. Silva, Julien Valton.
Application Number | 20130117869 13/440940 |
Document ID | / |
Family ID | 45976522 |
Filed Date | 2013-05-09 |
United States Patent
Application |
20130117869 |
Kind Code |
A1 |
DUCHATEAU; PHILIPPE ; et
al. |
May 9, 2013 |
METHOD FOR THE GENERATION OF COMPACT TALE-NUCLEASES AND USES
THEREOF
Abstract
The present invention relates to a method for the generation of
compact Transcription Activator-Like Effector Nucleases (TALENs)
that can efficiently target and process double-stranded DNA. More
specifically, the present invention concerns a method for the
creation of TALENs that consist of a single TALE DNA binding domain
fused to at least one catalytic domain such that the active entity
is composed of a single polypeptide chain for simple and efficient
vectorization and does not require dimerization to target a
specific single double-stranded DNA target sequence of interest and
process DNA nearby said DNA target sequence. The present invention
also relates to compact TALENs, vectors, compositions and kits used
to implement the method.
Inventors: |
DUCHATEAU; PHILIPPE;
(Gargan, FR) ; Valton; Julien; (Paris, FR)
; Bertonati; Claudia; (Paris, FR) ; Epinat;
Jean-Charles; (Paris, FR) ; Silva; George H.;
(Trevise, FR) ; Juillerat; Alexandre; (Paris,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DUCHATEAU; PHILIPPE
Valton; Julien
Bertonati; Claudia
Epinat; Jean-Charles
Silva; George H.
Juillerat; Alexandre |
Gargan
Paris
Paris
Paris
Trevise
Paris |
|
FR
FR
FR
FR
FR
FR |
|
|
Assignee: |
Cellectis S.A.
Romainville Cedex
FR
|
Family ID: |
45976522 |
Appl. No.: |
13/440940 |
Filed: |
April 5, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61472065 |
Apr 5, 2011 |
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61496454 |
Jun 13, 2011 |
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61499043 |
Jun 20, 2011 |
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61499047 |
Jun 20, 2011 |
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61533098 |
Sep 9, 2011 |
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61533123 |
Sep 9, 2011 |
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61579544 |
Dec 22, 2011 |
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Current U.S.
Class: |
800/13 ;
424/94.6; 435/196; 435/320.1; 435/358; 435/419; 435/440; 435/91.53;
536/23.2; 800/298 |
Current CPC
Class: |
C07K 2319/07 20130101;
C07K 2319/00 20130101; C12N 9/22 20130101; A61P 43/00 20180101;
A61K 38/00 20130101; C12N 9/16 20130101; C12P 19/34 20130101; C07K
2319/80 20130101; C07K 14/4703 20130101 |
Class at
Publication: |
800/13 ;
435/91.53; 435/196; 435/320.1; 435/358; 800/298; 435/440; 435/419;
536/23.2; 424/94.6 |
International
Class: |
C12N 9/16 20060101
C12N009/16 |
Claims
1) A method for targeting and processing a double-stranded DNA,
comprising: (a) selecting one DNA target sequence of interest on
one strand of a double-stranded DNA; (b) providing a unique compact
TALEN monomer comprising: (i) one core TALE scaffold comprising
Repeat Variable Dipeptide regions (RVDs) having DNA binding
specificity onto said DNA target sequence of interest; (ii) at
least one catalytic domain wherein said catalytic domain is capable
of processing DNA a few base pairs away from said DNA target
sequence of interest when fused to the C and/or N terminal of said
core TALE scaffold from (i); (iii) optionally one peptidic linker
to fuse said catalytic domain from (ii) to said core TALE scaffold
from (i) when needed; wherein said compact TALEN monomer is
assembled to bind and process said double stranded DNA without
requiring dirnerization; (c) contacting said double-stranded DNA
with said unique monomer such that the double-stranded is processed
a few base pairs away in 3' and/or 5' direction(s) from said one
strand target sequence.
2) A method according to claim 1, wherein said catalytic domain has
cleavage activity on said double-stranded DNA.
3) A method according to claim 1, wherein said catalytic domain is
fused to the C-terminal domain of said core TALE scaffold.
4) A method according to claim 1, wherein said catalytic domain is
fused to the N-terminal domain of said core TALE scaffold.
5) A method according to claim 1, wherein one catalytic domain is
fused to the C-terminal domain and another catalytic domain is
fused to the N-terminal domain of said core TALE scaffold.
6) A method according to claim 1, wherein said catalytic domain is
selected from the group consisting of proteins listed in Table 2 or
a functional mutant thereof.
7) A method according to claim 1, wherein said catalytic domain is
I-TevI (SEQ ID NO: 20) or a functional mutant thereof.
8) A method according to claim 7, wherein I-TevI (SEQ ID NO: 20) or
said functional mutant thereof is fused to the N-terminal domain of
said core TALE scaffold.
9) A method according to claim 8, comprising a protein sequence
having at least 80%, more preferably 90%, again more preferably 95%
amino acid sequence identity with the protein sequences selected
from the group of SEQ ID NO: 426-432.
10) A method according to claim 1, wherein said catalytic domain is
ColE7 (SEQ ID NO: 11) or a functional mutant thereof.
11) A method according to claim 10, wherein ColE7 (SEQ ID NO: 11)
or said functional mutant thereof is fused to the C-terminus part
of said core TALE scaffold.
12) A method according to claim 10, wherein ColE7 (SEQ ID NO: 11)
or said functional mutant thereof is fused to the N-terminus part
of said core TALE scaffold.
13) A method according to claim 11, comprising a protein sequence
having at least 80%, more preferably 90%, again more preferably 95%
amino acid sequence identity with the protein sequences selected
from the group of SEQ ID NO: 435-438.
14) A method according to claim 1, wherein said catalytic domain is
NucA (SEQ ID NO: 26) or a functional mutant thereof.
15) A method according to claim 14, wherein NucA (SEQ ID NO: 26) or
said functional mutant thereof is fused to the C-terminus part of
said core TALE scaffold.
16) A method according to claim 14, wherein NucA (SEQ ID NO: 26) or
said functional mutant thereof is fused to the N-terminus part of
said core TALE scaffold.
17) A method according to claim 15, comprising a protein sequence
having at least 80%, more preferably 90%, again more preferably 95%
amino acid sequence identity with the protein sequences selected
from the group of SEQ ID NO: 433-434.
18) A method according to claim 1, wherein said catalytic domain is
I-CreI (SEQ ID NO: 1) or a functional mutant thereof.
19) A method according to claim 18, wherein I-CreI (SEQ ID NO: 1)
or said functional mutant thereof is fused to the C-terminus part
of said core TALE scaffold.
20) A method according to claim 18, wherein I-CreI (SEQ ID NO: 1)
or said functional mutant thereof is fused to the N-terminus part
of said core TALE scaffold.
21) A method according to claim 19, comprising a protein sequence
having at least 80%, more preferably 90%, again more preferably 95%
amino acid sequence identity with the protein sequences selected
from the group of SEQ ID NO: 439-441 and SEQ ID NO: 444-446.
22) A method according to claim 1, wherein said core TALE scaffold
comprises a protein sequence having at least 80%, more preferably
90%, again more preferably 95% amino acid sequence identity with
the protein sequences selected from the group consisting of SEQ ID
NO: 134 and SEQ ID NO: 135.
23) A method according to claim 1, wherein said core TALE scaffold
comprises a protein sequence having at least 80%, more preferably
90%, again more preferably 95% amino acid sequence identity with
the protein sequences selected from the group consisting of SEQ ID
NO: 136 to SEQ ID NO: 139.
24) A method according to claim 1, wherein said unique compact
TALEN monomer further comprises: (i) at least one enhancer domain;
(ii) Optionally one peptide linker to fuse said enhancer domain to
one part of said unique compact TALEN monomer active entity.
25) A method according to claim 1, wherein said peptidic linker
sequence can be selected from the group consisting of SEQ ID NO:
67-104 and SEQ ID NO: 372 to SEQ ID NO: 415.
26) A method according to claim 5, wherein said unique compact
TALEN monomer comprises a combination of two catalytic domains
respectively fused to the C-terminus part and to the N-terminus
part of said core TALE scaffold selected from the group consisting
of: (i) A Nuc A domain (SEQ ID NO: 26) in N-terminus and a Nuc A
domain (SEQ ID NO: 26) in C-terminus; (ii) A ColE7 domain (SEQ ID
NO: 11) in N-terminus and a ColE7 domain (SEQ ID NO: 11) in
C-terminus; (iii) A TevI domain (SEQ ID NO: 20) in N-terminus and a
ColE7 domain (SEQ ID NO: 11) in C-terminus; (iv) A TevI domain (SEQ
ID NO: 20) in N-terminus and a NucA domain (SEQ ID NO: 26) in
C-terminus; (v) A ColE7 domain (SEQ ID NO: 11) in N-terminus and a
NucA domain (SEQ ID NO: 26) in C-terminus; (vi) A NucA domain (SEQ
ID NO: 26) in N-terminus and a ColE7 domain (SEQ ID NO: 11) in
C-terminus.
27) A method according to claim 29, comprising a protein sequence
having at least 80%, more preferably 90%, again more preferably 95%
amino acid sequence identity with the protein sequences selected
from the group consisting of SEQ ID NO: 448 and 450.
28) A method according to claim 5, wherein said unique compact
TALEN monomer comprises a combination of two catalytic domains
respectively fused to the C-terminus part and to the N-terminus
part of said core TALE scaffold selected from the group consisting
of: (i) A TevI domain (SEQ ID NO: 20) in N-terminus and a FokI
domain (SEQ ID NO: 368) in C-terminus; (ii) A TevI domain (SEQ ID
NO: 20) in N-terminus and a TevI domain (SEQ ID NO: 20) in
C-terminus; (iii) A scTrex2 domain (SEQ ID NO: 451) in N-terminus
and a FokI domain (SEQ ID NO: 368) in C-terminus.
29) A method according to claim 28, comprising a protein sequence
having at least 80%, more preferably 90%, again more preferably 95%
amino acid sequence identity with the protein sequences selected
from the group consisting of SEQ ID NO: 447-450 and SEQ ID NO:
452.
30) A compact TALEN monomer comprising: (i) one core TALE scaffold
comprising Repeat Variable Dipeptide regions (RVDs) having DNA
binding specificity onto a specific double-stranded DNA target
sequence of interest; (ii) at least one catalytic domain wherein
said catalytic domain is capable of processing DNA a few base pairs
away from said double-stranded DNA target sequence of interest when
fused to the C or N terminal of said core TALE scaffold from (i);
(iii) optionally one peptidic linker to fuse said catalytic domain
from (ii) to said engineered core TALE scaffold from (i) when
needed; wherein said compact TALEN monomer is assembled to bind
said target DNA sequence and process double-stranded DNA without
requiring dimerization.
31) A compact TALEN monomer according to claim 30, wherein said
catalytic domain has cleavage activity on the double-stranded
DNA.
32) A compact TALEN monomer according to claim 30, wherein said
catalytic domain is fused to the C-terminal domain of said core
TALE scaffold.
33) A compact TALEN monomer according to claim 30, wherein said
catalytic domain is fused to the N-terminal domain of said core
TALE scaffold.
34) A compact TALEN monomer according to claim 30, wherein one
catalytic domain is fused to the C-terminal domain and another
catalytic domain is fused to the N-terminal domain of said core
TALE scaffold.
35) A compact TALEN monomer according to claim 30, wherein said
catalytic domain is selected from the group consisting of proteins
listed in Table 2 or a functional mutant thereof.
36) A compact TALEN monomer according to claim 30, wherein said
catalytic domain is I-TevI (SEQ ID NO: 20) or a functional mutant
thereof.
37) A compact TALEN monomer according to claim 36, wherein I-TevI
(SEQ ID NO: 20) or said functional mutant thereof is fused to the
N-terminal domain of said core TALE scaffold.
38) A compact TALEN monomer according to claim 37, comprising a
protein sequence having at least 80%, more preferably 90%, again
more preferably 95% amino acid sequence identity with the protein
sequences selected from the group of SEQ ID NO: 426-432.
39) A compact TALEN monomer according to claim 30, wherein said
catalytic domain is ColE7 (SEQ ID NO: 11) or a functional mutant
thereof.
40) A compact TALEN monomer according to claim 39, wherein ColE7
(SEQ ID NO: 11) or said functional mutant thereof is fused to the
C-terminus part of said core TALE scaffold.
41) A compact TALEN monomer according to claim 39, wherein ColE7
(SEQ ID NO: 11) or said functional mutant thereof is fused to the
N-terminus part of said core TALE scaffold.
42) A compact TALEN monomer according to claim 40, comprising a
protein sequence having at least 80%, more preferably 90%, again
more preferably 95% amino acid sequence identity with the protein
sequences selected from the group of SEQ ID NO:435-438.
43) A compact TALEN monomer according to claim 30, wherein said
catalytic domain is NucA (SEQ ID NO: 26) or a functional mutant
thereof.
44) A compact TALEN monomer according to claim 43, wherein NucA
(SEQ ID NO: 26) or said functional mutant thereof is fused to the
C-terminus part of said core TALE scaffold.
45) A compact TALEN monomer according to claim 43, wherein NucA
(SEQ ID NO: 26) or said functional mutant thereof is fused to the
N-terminus part of said core TALE scaffold.
46) A compact TALEN monomer according to claim 44, comprising a
protein sequence having at least 80%, more preferably 90%, again
more preferably 95% amino acid sequence identity with the protein
sequences selected from the group of SEQ ID NO:433-434.
47) A compact TALEN monomer according to claim 30, wherein said
catalytic domain is I-CreI (SEQ ID NO: 1) or a functional mutant
thereof.
48) A compact TALEN monomer according to claim 47, wherein I-CreI
(SEQ ID NO: 1) or said functional mutant thereof is fused to the
C-terminus part of said core TALE scaffold.
49) A compact TALEN monomer according to claim 47, wherein I-CreI
(SEQ ID NO: 1) or said functional mutant thereof is fused to the
N-terminus part of said core TALE scaffold.
50) A compact TALEN monomer according to claim 48, comprising a
protein sequence having at least 80%, more preferably 90%, again
more preferably 95% amino acid sequence identity with the protein
sequences selected from the group of SEQ ID NO: 444-446.
51) A compact TALEN monomer according to claim 30, wherein said
core TALE scaffold comprises a protein sequence having at least
80%, more preferably 90%, again more preferably 95% amino acid
sequence identity with the protein sequences selected from the
group consisting of SEQ ID NO: 134 and SEQ ID NO: 135.
52) A compact TALEN monomer according to claim 30, wherein said
core TALE scaffold comprises a protein sequence having at least
80%, more preferably 90%, again more preferably 95% amino acid
sequence identity with the protein sequences selected from the
group consisting of SEQ ID NO: 136 to SEQ ID NO: 139.
53) A compact TALEN monomer according to claim 30, wherein said
unique compact TALEN monomer further comprises: (i) At least one
enhancer domain; (ii) Optionally one peptide linker to fuse said
enhancer domain to one part of said unique compact TALEN monomer
active entity.
54) A compact TALEN monomer according to claim 30, wherein said
peptidic linker sequence can be selected from the group consisting
of SEQ ID NO: 67-104 and SEQ ID NO: 372 to SEQ ID NO: 415.
55) A compact TALEN monomer according to claim 34, comprising a
combination of two catalytic domains respectively fused to the
C-terminus part and to the N-terminus part of said core TALE
scaffold selected from the group consisting of: (i) A Nuc A domain
(SEQ ID NO: 26) in N-terminus and a Nuc A domain (SEQ ID NO: 26) in
C-terminus; (ii) A ColE7 domain (SEQ ID NO: 11) in N-terminus and a
ColE7 domain (SEQ ID NO: 11) in C-terminus; (iii) A TevI domain
(SEQ ID NO: 20) in N-terminus and a ColE7 domain (SEQ ID NO: 11) in
C-terminus; (iv) A TevI domain (SEQ ID NO: 20) in N-terminus and a
NucA domain (SEQ ID NO: 26) in C-terminus; (v) A ColE7 domain (SEQ
ID NO: 11) in N-terminus and a NucA domain (SEQ ID NO: 26) in
C-terminus; (vi) A NucA domain (SEQ ID NO: 26) in N-terminus and a
ColE7 domain (SEQ ID NO: 11) in C-terminus.
56) A compact TALEN monomer according to claim 55, comprising a
protein sequence having at least 80%, more preferably 90%, again
more preferably 95% amino acid sequence identity with the protein
sequences selected from the group consisting of SEQ ID NO: 448 and
450.
57) A compact TALEN monomer according to claim 37, wherein said
unique compact TALEN monomer comprises a combination of two
catalytic domains respectively fused to the C-terminus part and to
the N-terminus part of said core TALE scaffold selected from the
group consisting of: (i) A TevI domain (SEQ ID NO: 20) in
N-terminus and a FokI domain (SEQ ID NO: 368) in C-terminus; (ii) A
TevI domain (SEQ ID NO: 20) in N-terminus and a TevI domain (SEQ ID
NO: 20) in C-terminus; (iii) A scTrex2 domain (SEQ ID NO: 451) in
N-terminus and a FokI domain (SEQ ID NO: 368) in C-terminus.
58) A method according to claim 63, comprising a protein sequence
having at least 80%, more preferably 90%, again more preferably 95%
amino acid sequence identity with the protein sequences selected
from the group consisting of SEQ ID NO: 447-450 and SEQ ID NO:
452.
59) A recombinant polynucleotide encoding a compact TALEN according
to any one of claims 30 to 58.
60) A vector comprising a recombinant polynucleotide according to
claim 59.
61) A composition comprising a compact TALEN according to claim 30
and a carrier.
62) A pharmaceutical composition comprising a compact TALEN
according to claim 30 and a pharmaceutically active carrier.
63) A host cell which comprises a recombinant polynucleotide of
claim 59.
64) A non-human transgenic animal which comprises a recombinant
polynucleotide of claim 59.
65) A non-human transgenic animal which comprises a vector of claim
60.
66) A transgenic plant which comprises a recombinant polynucleotide
of claim 59.
67) A transgenic plant which comprises a vector of claim 60.
68) A kit comprising a compact TALEN monomer according to claim 30
and instructions for use in enhancing DNA processing efficiency of
a single double-stranded DNA target sequence of interest.
69) A method for increasing targeted Homologous Recombination
comprising a compact TALEN monomer according to claim 30wherein at
least one catalytic domain has a cleavase activity.
70) A method for increasing targeted Homologous Recombination with
less Non Homologous End-joining comprising a compact TALEN monomer
according to claim 30 wherein at least one catalytic domain has a
nickase activity.
71) A method for increasing excision of a single-strand of DNA
spanning the binding region of a compact TALEN monomer according to
claim 30 wherein: (i) at least one catalytic domain has a cleavase
activity; (ii) at least one catalytic domain has a nickase
activity.
72) A method of treatment of a genetic disease caused by a mutation
in a specific single double-stranded DNA target sequence in a gene
comprising administering to a subject in need thereof an effective
amount of a compact TALEN of claim 30 or a variant thereof.
73) A method for inserting a transgene into a specific single
double-stranded DNA target sequence of a genomic locus of a cell,
tissue or non-human animal wherein at least one compact TALEN
monomer of claim 30 is introduced in said cell, tissue or non-human
animal.
74) A method to modulate the activity of a compact TALEN monomer
according to claim 30 when expressed in a cell wherein said method
comprises the step of introducing in said cell an auxiliary domain
modulating the activity of said compact TALEN.
75) A method according to claim 74 to inhibit the activity of a
compact TALEN monomer comprising: (iv) one core TALE scaffold
comprising Repeat Variable Dipeptide regions (RVDs) having DNA
binding specificity onto a specific double-stranded DNA target
sequence of interest; (v) at least one catalytic domain wherein
said catalytic domain is capable of processing DNA a few base pairs
away from said double-stranded DNA target sequence of interest when
fused to the C or N terminal of said core TALE scaffold from (i);
(vi) optionally one peptidic linker to fuse said catalytic domain
from (ii) to said engineered core TALE scaffold from (i) when
needed; wherein said compact TALEN monomer is assembled to bind
said target DNA sequence and process double-stranded DNA without
requiring dimerization
76) A method according to claim 74 wherein the catalytic domain of
said compact TALEN monomer is NucA (SEQ ID NO: 26) and said
auxiliary domain is NuiA (SEQ ID NO: 229) or a functional mutant
thereof.
77) A method according to claim 74 wherein the catalytic domain of
said compact TALEN monomer is ColE7 (SEQ ID NO: 11) and said
auxiliary domain is Im7 (SEQ ID NO: 230) or a functional mutant
thereof.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method for the generation
of compact Transcription Activator-Like Effector Nucleases (TALENs)
that can efficiently target and process double-stranded DNA. More
specifically, the present invention concerns a method for the
creation of TALENs that consist of a single TALE DNA binding domain
fused to at least one catalytic domain such that the active entity
is composed of a single polypeptide chain for simple and efficient
vectorization and does not require dimerization to target a
specific single double-stranded DNA target sequence of interest and
process DNA nearby said DNA target sequence. The present invention
also relates to compact TALENs, vectors, compositions and kits used
to implement the method.
BACKGROUND OF THE INVENTION
[0002] Mammalian genomes constantly suffer from various types of
damage, of which double-strand breaks (DSBs) are considered the
most dangerous (Haber 2000). Repair of DSBs can occur through
diverse mechanisms that can depend on cellular context. Repair via
homologous recombination (HR) is able to restore the original
sequence at the break. Because of its strict dependence on
extensive sequence homology, this mechanism is suggested to be
active mainly during the S and G2 phases of the cell cycle where
the sister chromatids are in close proximity (Sonoda, Hochegger et
al. 2006). Single-strand annealing (SSA) is another
homology-dependent process that can repair DSBs between direct
repeats and thereby promotes deletions (Paques and Haber 1999).
Finally, non-homologous end joining (NHEJ) of DNA is a major
pathway for the repair of DSBs that can function throughout the
cell cycle and does not depend on homologous recombination (Moore
and Haber 1996; Haber 2008). NHEJ seems to comprise at least two
different components: (i) a pathway that consists mostly in the
direct re-joining of DSB ends, and which depends on the XRCC4, Lig4
and Ku proteins, and; (ii) an alternative NHEJ pathway, which does
not depend on XRCC4, Lig4 and Ku, and is especially error-prone,
resulting mostly in deletions, with the junctions occurring between
micro-homologies (Frank, Sekiguchi et al. 1998; Gao, Sun et al.
1998; Guirouilh-Barbat, Huck et al. 2004; Guirouilh-Barbat, Rass et
al. 2007; Haber 2008; McVey and Lee 2008).
[0003] Homologous gene targeting (HGT), first described over 25
years ago (Hinnen, Hicks et al. 1978; Orr-Weaver, Szostak et al.
1981; Orr-Weaver, Szostak et al. 1983; Rothstein 1983), was one of
the first methods for rational genome engineering and remains to
this day a standard for the generation of engineered cells or
knock-out mice (Capecchi 2001). An inherently low efficiency has
nevertheless prevented it from being used as a routine protocol in
most cell types and organisms. To address these issues, an
extensive assortment of rational approaches has been proposed with
the intent of achieving greater than 1% targeted modifications.
Many groups have focused on enhancing the efficacy of HGT, with two
major disciplines having become apparent: (i) so-called "matrix
optimization" methods, essentially consisting of modifying the
targeting vector structure to achieve maximal efficacy, and; (ii)
methods involving additional effectors to stimulate HR, generally
sequence-specific endonucleases. The field of matrix optimization
has covered a wide range of techniques, with varying degrees of
success (Russell and Hirata 1998; Inoue, Dong et al. 2001; Hirata,
Chamberlain et al. 2002; Taubes 2002; Gruenert, Bruscia et al.
2003; Sangiuolo, Scaldaferri et al. 2008; Bedayat, Abdolmohamadi et
al. 2010). Stimulation of HR via nucleases, on the other hand, has
repeatedly proven efficient (Paques and Duchateau 2007; Carroll
2008).
[0004] For DSBs induced by biological reagents, e.g. meganucleases,
ZFNs and TALENs (see below), which cleave DNA by hydrolysis of two
phosphodiester bonds, the DNA can be rejoined in a seamless manner
by simple re-ligation of the cohesive ends. Alternatively,
deleterious insertions or deletions (indels) of various sizes can
occur at the breaks, eventually resulting in gene inactivation
(Liang, Han et al. 1998; Lloyd, Plaisier et al. 2005; Doyon,
McCammon et al. 2008; Perez, Wang et al. 2008; Santiago, Chan et
al. 2008; Kim, Lee et al. 2009; Yang, Djukanovic et al. 2009). The
nature of this process, which does not rely on site-specific or
homologous recombination, gives rise to a third targeted approach
based on endonuclease-induced mutagenesis. This approach, as well
as the related applications, may be simpler than those based on
homologous recombination in that (a) one does not need to introduce
a repair matrix, and; (b) efficacy will be less cell-type dependant
(in contrast to HR, NHEJ is probably active throughout the cell
cycle (Delacote and Lopez 2008). Targeted mutagenesis based on NEHJ
has been used to trigger inactivation of single or even multiple
genes in immortalized cell lines (Cost, Freyvert et al. 2010; Liu,
Chan et al. 2010). In addition, this method opens new perspectives
for organisms in which the classical HR-based gene knock-out
methods have proven inefficient, or at least difficult to establish
(Doyon, McCammon et al. 2008; Geurts, Cost et al. 2009; Shukla,
Doyon et al. 2009; Yang, Djukanovic et al. 2009; Gao, Smith et al.
2010; Mashimo, Takizawa et al. 2010; Menoret, Iscache et al.
2010).
[0005] Over the last 15 years, the use of meganucleases to
successfully induce gene targeting has been well documented,
starting from straightforward experiments involving wild-type
I-SceI to more refined work involving completely re-engineered
enzymes (Stoddard, Scharenberg et al. 2007; Galetto, Duchateau et
al. 2009; Marcaida, Munoz et al. 2010; Arnould, Delenda et al.
2011). Meganucleases, also called homing endonucleases (HEs), can
be divided into five families based on sequence and structure
motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box and PD-(D/E)XK
(Stoddard 2005; Zhao, Bonocora et al. 2007). Structural data are
available for at least one member of each family. The most well
studied family is that of the LAGLIDADG proteins, with a
considerable body of biochemical, genetic and structural work
having established that these endonucleases could be used as
molecular tools (Stoddard, Scharenberg et al. 2007; Arnould,
Delenda et al. 2011). Member proteins are composed of domains that
adopt a similar .alpha..beta..beta..alpha..beta..beta..alpha. fold,
with the LAGLIDADG motif comprising the terminal region of the
first helix and not only contributing to a bipartite catalytic
center but also forming the core subunit/subunit interaction
(Stoddard 2005). Two such .alpha./.beta. domains assemble to form
the functional protein, with the .beta.-strands in each creating a
saddle-shaped DNA binding region. The spatial separation of the
catalytic center with regions directly interacting with the DNA has
allowed for specificity re-engineering (Seligman, Chisholm et al.
2002; Sussman, Chadsey et al. 2004; Arnould, Chames et al. 2006;
Doyon, Pattanayak et al. 2006; Rosen, Morrison et al. 2006; Smith,
Grizot et al. 2006; Arnould, Perez et al. 2007). In addition,
whereas all known LAGLIDADG proteins analyzed to date act as
"cleavases" to cut both strands of the target DNA, recent progress
has been made in generating "mega-nickases" that cleave only one
strand (Niu, Tenney et al. 2008; McConnell Smith, Takeuchi et al.
2009). Such enzymes can in principle provide similar levels of
targeted induced HR with a minimization in the frequency of
NHEJ.
[0006] Although numerous engineering efforts have focused on
LAGLIDADG HEs, members from two other families, GIY-YIG and HNH,
are of particular interest. Biochemical and structural studies have
established that in both families, member proteins can adopt a
bipartite fold with distinct functional domains: (1) a catalytic
domain responsible mainly for DNA cleavage, and; (2) a DNA-binding
domain to provide target specificity (Stoddard 2005; Marcaida,
Munoz et al. 2010). The related GIY-YIG HEs I-TevI and I-BmoI have
been exploited to demonstrate the interchangeability of the
DNA-binding region for these enzymes (Liu, Derbyshire et al. 2006).
Analysis of the I-BasI HE revealed that although the N-terminal
catalytic domain belongs to the HNH family, the C-terminal
DNA-binding region resembles the intron-encoded endonuclease repeat
motif (IENR1) found in endonucleases of the GIY-YIG family
(Landthaler and Shub 2003). The catalytic head of I-BasI has
sequence similarity to those of the HNH HEs I-HmuI, I-HmuII and
I-TwoI, all of which function as strand-specific nickases
(Landthaler, Begley et al. 2002; Landthaler and Shub 2003;
Landthaler, Lau et al. 2004; Shen, Landthaler et al. 2004;
Landthaler, Shen et al. 2006).
[0007] Whereas the above families of proteins contain
sequence-specific nucleases, the HNH motif has also been identified
in nonspecific nucleases such the E. coli colicins (e.g. ColE9 and
ColE7), EndA from S. pneumoniae, NucA from Anabaena and CAD (Midon,
Schafer et al. 2011). As well as having the HNH motif, several of
these nucleases contain the signature DRGH motif and share
structural homology with core elements forming the
.beta..beta..alpha.-Me-finger active site motif. Mutational studies
of residues in the HNH/DRGH motifs have confirmed their role in
nucleic acid cleavage activity (Ku, Liu et al. 2002; Doudeva, Huang
et al. 2006; Eastberg, Eklund et al. 2007; Huang and Yuan 2007).
Furthermore, the DNA binding affinity and sequence preference for
ColE7 could be effectively altered (Wang, Wright et al. 2009). Such
detailed studies illustrate the potential in re-engineering
nonspecific nucleases for targeted purposes.
[0008] Zinc-finger nucleases (ZFNs), generated by fusing
Zinc-finger-based DNA-binding domains to an independent catalytic
domain via a flexible linker (Kim, Cha et al. 1996; Smith, Berg et
al. 1999; Smith, Bibikova et al. 2000), represent another type of
engineered nuclease commonly used to stimulate gene targeting. The
archetypal ZFNs are based on the catalytic domain of the Type IIS
restriction enzyme Fokl and have been successfully used to induce
gene correction, gene insertion, and gene deletion. Zinc
Finger-based DNA binding domains are made of strings of 3 or 4
individual Zinc Fingers, each recognizing a DNA triplet (Pabo,
Peisach et al. 2001). In theory, one of the major advantages of
ZFNs is that they are easy to design, using combinatorial assembly
of preexisting Zinc Fingers with known recognition patterns (Choo
and Klug 1994; Choo and Klug 1994; Kim, Lee et al. 2009). However,
close examination of high resolution structures shows that there
are actually cross-talks between units (Elrod-Erickson, Rould et
al. 1996), and several methods have been used to assemble ZF
proteins by choosing individual Zinc Fingers in a context dependant
manner (Greisman and Pabo 1997; Isalan and Choo 2001; Maeder,
Thibodeau-Beganny et al. 2008; Ramirez, Foley et al. 2008) to
achieve better success rates and reagents of better quality.
[0009] Recently, a new class of chimeric nuclease using a FokI
catalytic domain has been described (Christian, Cermak et al. 2010;
Li, Huang et al. 2011). The DNA binding domain of these nucleases
is derived from Transcription Activator Like Effectors (TALE), a
family of proteins used in the infection process by plant pathogens
of the Xanthomonas genus. In these DNA binding domains, sequence
specificity is driven by a series of 33-35 amino acids repeats,
differing essentially by two positions (Boch, Scholze et al. 2009;
Moscou and Bogdanove 2009). Each base pair in the DNA target is
contacted by a single repeat, with the specificity resulting from
the two variant amino acids of the repeat (the so-called repeat
variable dipeptide, RVD). The apparent modularity of these DNA
binding domains has been confirmed to a certain extent by modular
assembly of designed TALE-derived protein with new specificities
(Boch, Scholze et al. 2009; Moscou and Bogdanove 2009). However,
one cannot yet rule out a certain level of context dependence of
individual repeat/base recognition patterns, as was observed for
Zinc Finger proteins (see above). Furthermore, it has been shown
that natural TAL effectors can dimerize (Gurlebeck, Szurek et al.
2005) and how this would affect a "dimerization-based" TALE-derived
nuclease is currently unknown.
[0010] The functional layout of a FokI-based TALE-nuclease (TALEN)
is essentially that of a ZFN, with the Zinc-finger DNA binding
domain being replaced by the TALE domain (Christian, Cermak et al.
2010; Li, Huang et al. 2011). As such, DNA cleavage by a TALEN
requires two DNA recognition regions flanking an unspecific central
region. This central "spacer" DNA region is essential to promote
catalysis by the dimerizing FokI catalytic domain, and extensive
effort has been placed into optimizing the distance between the DNA
binding sites (Christian, Cermak et al. 2010; Miller, Tan et al.
2011). The length of the spacer has been varied from 14 to 30 base
pairs, with efficiency in DNA cleavage being interdependent with
spacer length as well as TALE scaffold construction (i.e. the
nature of the fusion construct used). It is still unknown whether
differences in the repeat region (i.e. RVD type and number used)
have an impact on the DNA "spacer" requirements or on the
efficiency of DNA cleavage by TALENs. Nevertheless, TALE-nucleases
have been shown to be active to various extents in cell-based
assays in yeast, mammalian cells and plants (Christian, Cermak et
al. 2010; Li, Huang et al. 2011; Mahfouz, Li et al. 2011; Miller,
Tan et al. 2011).
[0011] The inventors have developed a new type of TALEN that can be
engineered to specifically recognize and process target DNA
efficiently. These novel "compact TALENs" (cTALENs) do not require
dimerization for DNA processing activity, thereby alleviating the
need for "dual" target sites with intervening DNA "spacers".
Furthermore, the invention allows for generating several distinct
types of enzymes that can enhance separate DNA repair pathways (HR
vs. NHEJ).
BRIEF SUMMARY OF THE INVENTION
[0012] The present invention relates to a method to generate
compact Transcription Activator-Like Effector Nucleases (TALENs)
composed of a single polypeptide chain that do not require
dimerization to target a specific single double-stranded DNA target
sequence of interest and process DNA nearby said single
double-stranded DNA target sequence of interest. The present
invention also concerns the creation of functional single
polypeptide fusion proteins for simple and efficient vectorization.
In another aspect, the present invention relates to compact TALENs
comprising at least an enhancer domain wherein said enhancer domain
enhances the DNA processing efficiency of said compact TALENS
nearby a single double-stranded DNA target sequence of interest.
The present invention also relates to compact TALENS, vectors,
compositions and kits used to implement the method. The present
invention also relates to methods for use of said compact TALENs
according to the invention for various applications ranging from
targeted DNA cleavage to targeted gene regulation. The methods
according to the present invention can be used in various fields
ranging from the creation of transgenic organisms to treatment of
genetic diseases.
BRIEF DESCRIPTION OF THE FIGURES
[0013] In addition to the preceding features, the invention further
comprises other features which will emerge from the description
which follows, as well as to the appended drawings. A more complete
appreciation of the invention and many of the attendant advantages
thereof will be readily obtained as the same becomes better
understood by reference to the following Figures in conjunction
with the detailed description below.
[0014] FIG. 1: Endonuclease-induced gene targeting approaches. Upon
cleavage, DNA repair mechanisms may result in one of several
outcomes. (A) When a double-strand break is targeted between two
direct repeats, HR can result in the deletion of one repeat
together with the intervening sequence. Gene insertion (B) or
correction (C) can be achieved by the introduction of a DNA repair
matrix containing sequences homologous to the endogenous sequence
surrounding the DNA break. Mutations can be corrected either at or
distal to the break, with the frequency of correction decreasing
with increasing distance. (D) The misrepair of DNA ends by
error-prone NHEJ can result in insertions or deletions of various
sizes, leading to gene inactivation.
[0015] FIG. 2: Sequences of target DNA recognized by I-CreI and
I-TevI. C1234 (SEQ ID NO: 3) represents the partially symmetric
natural DNA sequence recognized and cleaved by the wild-type I-CreI
meganuclease. C1221 (SEQ ID NO: 2) represents an artificial
palindromic DNA sequence, derived from C1234 (SEQ ID NO: 3), also
recognized and cleaved by I-CreI (SEQ ID NO: 1). Nucleotides are
numbered outward (-/+) from the center of the target. DNA cleavage
occurs on either side of the underlined sequence to generate
4-nucleotide 3' overhanging ends. For I-CreI-based meganucleases,
the nature of the nucleotides at positions -2 to +2 can potentially
interfere with the cleavage activity of the protein. Tev (SEQ ID
NO: 105) represents the asymmetric DNA sequence recognized and
cleaved by the wild-type I-TevI meganuclease. Nucleotide numbering
is relative to the intron-insertion site of the natural target
sequence. Cleavage by I-TevI occurs on either side of the
underlined sequence to generate 2-nucleotide 3' overhanging
ends.
[0016] FIG. 3: Sequences of the target DNAs recognized by TALEN and
compact TALEN constructs. Target DNAs for the engineered compact
TALENS cTN-Avr and cTN-Pth are based on the naturally occurring
asymmetric sequences AvrBs3 (19 bp) [in bT1-Avr (SEQ ID NO: 136)
and bT2-Avr (SEQ ID NO: 137) baseline protein scaffolds] and PthXo1
(25 bp) [in bT1-Pth (SEQ ID NO: 138) and bT2-Pth (SEQ ID NO: 139)
baseline protein scaffolds], respectively. For each sequence,
nucleotides are numbered outward (-/+) from the anchoring T
(position -1). Sequences shown are directly contacted by the
protein to provide target specificity. Wild-type Repeat Variable
Dipeptides (RVDs) correspond to the dipeptides found in the repeats
of the naturally occurring effector proteins targeted to each
sequence. Cipher RVDs are based on the subset of
dipeptide/nucleotide pairs listed. Artificial RVDs are derived by
direct readout of the underlying DNA sequence using the cipher RVD
code (SEQ ID NO: 245 to 249).
[0017] FIG. 4: Schematic of meganuclease fusion configurations.
Fusion constructs are optimized to address or overcome distinct
problems. (A) The addition of two catalytic domains to an active
meganuclease can not only enhance cleavage activity (e.g. three
chances to effect DNA cleavage per binding event) but can also
promote sequence alterations by error-prone NHEJ since small
sections of DNA are excised for each pair of cleavage events. (B)
When specificity reengineering precludes maintaining cleavage
activity of the meganuclease, the attached catalytic domains
provide the necessary strand cleavage function. (C) and (D)
represent instances of (A) and (B), respectively, when only one
catalytic domain is tolerated per fusion protein (e.g. either as an
N- or C-terminal fusion or in the context of a single-chain
molecule). In all cases, the catalytic domain envisioned can be
either a cleavase (ability to cleave both strands of the DNA) or a
nickase (cleavage of only a single DNA strand) depending on the
application. Fusion junctions (N- vs. C-terminal) and linker
designs can vary with the application. Components of the fusion
proteins are listed in the legend.
[0018] FIG. 5: Schematic of cTALEN configurations. Compact TALENs
are designed to alleviate the need for multiple independent protein
moieties when targeting a DNA cleavage event. Importantly, the
requisite "spacer" region and dual target sites essential for the
function of current classical TALENs are unnecessary. In addition,
since the catalytic domain does not require specific DNA contacts,
there are no restrictions on regions surrounding the core TALE DNA
binding domain. (A) N-terminal fusion construct to promote HR via a
standard (cleavase domain) or conservative (nickase domain) repair
pathway. (B) C-terminal fusion construct with properties as in (A).
(C) The attachment of two catalytic domains to both ends of the
TALE allows for dual cleavage with enhancement in NHEJ. Fusion
junctions (N-vs. C-terminal) and linker designs can vary with the
application. Components of the fusion proteins are listed in the
legend.
[0019] FIG. 6: Schematic of enhanced cTALEN configurations. Compact
TALENs can be enhanced through the addition of a domain to promote
existing or alternate activities. As each end of the TALE DNA
binding domain is amenable to fusion, the order (N- v.s C-terminal)
of addition of the catalytic and enhancer domains can vary with the
application. (A) A standard cTALEN with a C-terminal enhancer
domain. (B) The enhancer domain is fused to the cTALEN via the
N-terminus of the catalytic domain. Such a configuration can be
used to assist and/or anchor the catalytic domain near the DNA to
increase cleavage activity. (C) The enhancer domain is sandwiched
between the catalytic domain and TALE DNA binding domain. The
enhancer domain can promote communication between the flanking
domains (i.e. to assist in catalysis and/or DNA binding) or can be
used to overcome the requisite T nucleotide at position -1 of all
TALE-based targets. (D) The enhancer domain is used to functionally
replace the natural TALE protein N-terminal region. (E) The
enhancer domain is used to functionally replace the natural TALE
protein C-terminal region. Fusion junctions (N-vs. C-terminal) and
linker designs can vary with the application. Components of the
fusion proteins are listed in the legend.
[0020] FIG. 7: Schematic of trans cTALEN configurations. Compact
TALENs can be combined with auxiliary enhancer domains to promote
alternate activities. Auxiliary domains provide an additional
function that is not essential to the cTALEN activity. (A) A
standard cTALEN with an N-terminal nickase catalytic domain becomes
a "cleavase" via the separate addition of the auxiliary domain. (B)
In some instances, the need to target the specificity of the
auxiliary domain is necessary. Such a configuration can achieved
via a TALE fusion and can be used to assist and/or anchor the
auxiliary domain near the DNA to increase activity of the cTALEN.
(C) The targeted auxiliary domain is provided either before or
after the cTALEN to perform an independent task. Communication
between the fusion proteins is not necessary. Fusion junctions
(N-vs. C-terminal) and linker designs can vary with the
application. Components of the fusion proteins are listed in the
legend.
[0021] FIG. 8: Schematic of DNA cleavage, in vivo re-ligation and
other repair pathways. In cells, cleavage by peptidic rare-cutting
endonucleases usually results in a DNA double strand break (DSB)
with cohesive ends. For example, meganucleases from the LAGLIDADG
family, such as I-SceI and I-CreI, produce DSBs with 3' overhangs.
These cohesive ends can be re-ligated in vivo by NHEJ, resulting in
seamless repair, and in the restoration of a cleavable target
sequence, which can in turn be processed again by the same
endonuclease. Thus, a series of futile cycles of cleavage and
re-ligation events can take place. Imprecise NHEJ or homologous
recombination can alter or remove the cleavage site, resulting in
cycle exit; this can also apply to compact TALENs and enhanced
compact TALENs according to the present invention (A). Two other
ways can also stop the process: (i) Chromosome loss can occur as
the consequence of failure to repair the DSB; (ii) a loss of
nuclease (degradation, dilution, cell division, etc. . . . ). B-E:
Consequences of cleavage of additional phosphodiester bonds. The
addition of a single nickase activity (B) or of two nickase
activities affecting the same strand (C) would result in a single
strand gap, and suppress the cohesive ends, which could in turn
affect the spectrum of events. Addition of two nickase activities
affecting opposite strands (D) or of a new cleavase activity
generating a second DSB (E) would result in a double strand gap; as
a consequence, perfect re-ligation is no longer possible, and one
or several alternative repair outcomes could be stimulated. The
current figure makes no assumption regarding the relative
frequencies of these alternative outcomes (imprecise NHEJ,
homologous recombination, others . . . ). Solid triangles represent
hydrolysis of phosphodiester bonds.
[0022] FIG. 9: Activity of TALE-AvrBs3::TevI in yeast (37.degree.
C.). The negative control consists in a TALEN without any RVDs.
n.d. indicates no detectable activity, +indicates an activity over
0.3 in yeast assay and +++indicates an activity over 0.7 in yeast
assay (International PCT Applications WO 2004/067736 and in Epinat,
Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et
al. 2006; Smith, Grizot et al. 2006).
[0023] FIG. 10: Activity of TALE-AvrBs3::TevI in mammalian cells.
(Extrachromosomic assay in CHO-K1). pCLS8993 (SEQ ID NO: 194) is
represented by a black bar and pCLS8994 (SEQ ID NO: 195) is
represented by a dark grey bar. Negative control (empty vector) by
a white bar and positive control (I-SceI meganuclease) by a light
grey bar. Data are normalized relative to the positive control.
[0024] FIG. 11: Activity of TALE-AvrBs3::NucA in yeast (37.degree.
C.). The negative control is a target lacking a recognition site
(neg. ctrl.: SEQ ID NO: 228). Compact is a target having only one
recognition site (SEQ ID NO: 224). n.d. indicates no detectable
activity, +indicates an activity over 0.3 in yeast assay at
37.degree. C.; ++, activity over 0.5 in yeast assay at 37.degree.
C. and +++activity over 0.7 in yeast assay at 37.degree. C.
(International PCT Applications WO 2004/067736 and in Epinat,
Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et
al. 2006; Smith, Grizot et al. 2006).
[0025] FIG. 12: Activity of TALE-AvrBs3::ColE7 in yeast (37.degree.
C.). The negative control is a target lacking a recognition site
(neg. ctrl.: SEQ ID NO: 228). Compact is a target having only one
recognition site (SEQ ID NO: 224). n.d. indicates no detectable
activity, +indicates an activity over 0.3 in yeast assay at
37.degree. C.; ++, activity over 0.5 in yeast assay at 37.degree.
C. and +++activity over 0.7 in yeast assay at 37.degree. C.
(International PCT Applications WO 2004/067736 and in Epinat,
Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et
al. 2006; Smith, Grizot et al. 2006).
[0026] FIG. 13: Schematic and details of a TALE::CreI prototypical
compact TALEN. This class of compact TALEN targets a bipartite
recognition sequence comprised of the TALE DNA binding site
proximal to a meganuclease target site. The engineered TCRB02-A
meganuclease site is shown along with details of the RVDs and DNA
sequences recognized by the TALE moiety. A region of the T cell
receptor B gene is presented, highlighting the endogenous layout of
the TALE::CreI-based compact TALEN hybrid target site.
[0027] FIG. 14: Activity of TALE::scTB2aD01-based constructs in
yeast (30.degree. C.). The layouts of the various hybrid targets
are shown, starting (5') with the region recognized by the TALE DNA
binding domain in uppercase, the unspecific spacer region in
lowercase and the meganuclease target site in underlined uppercase
characters. Activity in yeast is illustrated for select
representative constructs. n.d. indicates no detectable activity,
+indicates an activity over 0.3 in yeast assay at 30.degree. C.;
++, activity over 0.5 in yeast assay at 30.degree. C. and
+++activity over 0.7 in yeast assay at 30.degree. C. (International
PCT Applications WO 2004/067736 and in Epinat, Arnould et al. 2003;
Chames, Epinat et al. 2005; Arnould, Chames et al. 2006; Smith,
Grizot et al. 2006).
[0028] FIG. 15: Western blot of TALE::scTB2aD01-based constructs.
Constructs were expressed in HEK293 cells and total protein
extracts were prepared 48 hours post-transfection. Protein was
detected using a polyclonal anti-1-CreI antibody.
[0029] FIG. 16: Toxicity of TALE::scTB2aD01-based constructs in
CHOK1 cells. Cytotoxicty is based on detectable levels of GFP
expressed in living cells, on day 1 vs day 6, relative to a
standard control (transfection of empty plasmid).
[0030] FIG. 17: NHEJ activity of TALE::scTB2aD01-based constructs
in HEK293 cells. A post-transfection PCR-based analysis of genomic
DNA is used to assess activity in vivo. Cleavage of mismatched DNA
sequences by T7 endonuclease is indicative of NHEJ events resulting
from the activity of the cTALEN or meganuclease at the targeted
locus.
[0031] FIG. 18: Activity of TevI::TALE-AvrBs3
+/--TALE-RagT2-R::TevI in yeast (37.degree. C.). The negative
control is a target lacking a recognition site (neg. ctrl.: SEQ ID
NO: 228). n.d. indicates no detectable activity, +indicates an
activity over 0.3 in yeast assay at 37.degree. C.; ++, activity
over 0.5 in yeast assay at 37.degree. C. and +++activity over 0.7
in yeast assay at 37.degree. C. (International PCT Applications WO
2004/067736 and in Epinat, Arnould et al. 2003; Chames, Epinat et
al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al.
2006).
[0032] FIG. 19: Activity of TALE::SnaseSTAAU in yeast (37.degree.
C.). The negative control is a target lacking recognition sites.
Compact is a target having only one recognition site (SEQ ID NO:
224). n.d. indicates no detectable activity at 37.degree. C.,
+/-indicated an activity above 0.3 in yeast assay at 37.degree. C.;
+indicated an activity over 0.3 in yeast assay at 37.degree. C.;
++indicated an activity over 0.5 in yeast assay at 37.degree. C.;
+++indicated an activity over 0.75 in yeast assay at 37.degree. C.
(International PCT Applications WO 2004/067736 and in Epinat,
Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et
al. 2006; Smith, Grizot et al. 2006).
[0033] FIG. 20: Activity of TALENColE7 with various polypeptide
linker in yeast (37.degree. C.). Compact is a target having only
one recognition site (SEQ ID NO: 224). n.d. indicates no detectable
activity at 37.degree. C., +indicated an activity over 0.3 in yeast
assay at 37.degree. C.; ++indicated an activity over 0.5 in yeast
assay at 37.degree. C.; +++indicated an activity over 0.75 in yeast
assay at 37.degree. C. (International PCT Applications WO
2004/067736 and in Epinat, Arnould et al. 2003; Chames, Epinat et
al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al.
2006).
[0034] Table 6: List of AvrBs3 targets with various spacer lengths
(SEQ ID NO: 157 to 192).
[0035] Table 7: List of AvrBs3 targets with various spacer lengths
(SEQ ID NO: 157 to 192) including a target with only one
recognition site (compact, SEQ ID NO: 224) and a negative control
target (neg. ctrl., SEQ ID NO: 228) consisting in a target without
any recognition site.
[0036] Table 13: List of hybrid RagT2-R/AvrBs3 targets with various
spacer lengths (SEQ ID NO: 315 to 350).
DETAILED DESCRIPTION OF THE INVENTION
[0037] Unless specifically defined herein, all technical and
scientific terms used have the same meaning as commonly understood
by a skilled artisan in the fields of gene therapy, biochemistry,
genetics, and molecular biology.
[0038] All methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, with suitable methods and materials being
described herein. All publications, patent applications, patents,
and other references mentioned herein are incorporated by reference
in their entirety. In case of conflict, the present specification,
including definitions, will prevail. Further, the materials,
methods, and examples are illustrative only and are not intended to
be limiting, unless otherwise specified.
[0039] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology, transgenic biology, microbiology,
recombinant DNA, and immunology, which are within the skill of the
art. Such techniques are explained fully in the literature. See,
for example, Current Protocols in Molecular Biology (Frederick M.
AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA);
Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et
al, 2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory
Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et
al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D.
Harries & S. J. Higgins eds. 1984); Transcription And
Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of
Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987);
Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A
Practical Guide To Molecular Cloning (1984); the series, Methods In
ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press,
Inc., New York), specifically, Vols. 154 and 155 (Wu et al. eds.)
and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene
Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos
eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods
In Cell And Molecular Biology (Mayer and Walker, eds., Academic
Press, London, 1987); Handbook Of Experimental Immunology, Volumes
I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating
the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1986).
[0040] In a first aspect, the present invention relates to a method
to generate compact Transcription Activator-Like Effector Nucleases
(cTALENs) composed of a single polypeptide chain that do not
require dimerization to target a specific single double-stranded
DNA target sequence of interest and process DNA nearby said single
double-stranded DNA target sequence of interest.
[0041] According to a first aspect of the present invention is a
method to generate compact Transcription Activator-Like Effector
Nucleases (cTALENs) comprising the steps of: [0042] (i) Engineering
a core TALE scaffold (a) comprising different sets of Repeat
Variable Dipeptide regions (RVDs) to change DNA binding specificity
and target a specific single double-stranded DNA target sequence of
interest, (b) onto which a selection of catalytic domains can be
attached to effect DNA processing; [0043] (ii) Determining or
engineering at least one catalytic domain wherein said catalytic
domain is capable of processing DNA nearby said single
double-stranded DNA target sequence of interest when fused to said
engineered core TALE scaffold from (i); [0044] (iii) Optionally
determining or engineering a peptidic linker to fuse said catalytic
domain from (ii) to said engineered core TALE scaffold from (i);
thereby obtaining a compact TALEN entity composed of a single
polypeptide chain that does not require dimerization to target a
specific single double-stranded DNA target sequence of interest and
process DNA nearby said single double-stranded DNA target sequence
of interest. In other words, the compact TALEN according to the
present invention is an active entity unit able, by itself, to
target only one specific single double-stranded DNA target sequence
of interest through one DNA binding domain and to process DNA
nearby said single double-stranded DNA target sequence of
interest.
[0045] In another embodiment, is a method for targeting and
processing a double-stranded DNA, comprising: [0046] (a) Selecting
one DNA target sequence of interest on one strand of a
double-stranded DNA; [0047] (b) Providing a unique compact TALEN
monomer comprising: [0048] (i) One core TALE scaffold comprising
Repeat Variable Dipeptide regions (RVDs) having DNA binding
specificity onto said DNA target sequence of interest; [0049] (ii)
At least one catalytic domain wherein said catalytic domain is
capable of processing DNA a few base pairs away from said DNA
target sequence of interest when fused to the C and/or N terminal
of said core TALE scaffold from (i); [0050] (iii) Optionally one
peptidic linker to fuse said catalytic domain from (ii) to said
core TALE scaffold from (i) when needed; [0051] wherein said
compact TALEN monomer is assembled to bind and process said double
stranded DNA without requiring dimerization; [0052] (c) Contacting
said double-stranded DNA with said unique monomer such that the
double-stranded is processed a few base pairs away in 3' and/or 5'
direction(s) from said one strand target sequence.
[0053] In another embodiment, said engineered core TALE scaffold
according to the present invention comprises an additional
N-terminal domain resulting in an engineered core TALE scaffold
sequentially comprising a N-terminal domain and different sets of
Repeat Variable Dipeptide regions (RVDs) to change DNA binding
specificity and target a specific single double-stranded DNA target
sequence of interest, onto which a selection of catalytic domains
can be attached to effect DNA processing.
[0054] In another embodiment, said engineered core TALE scaffold
according to the present invention comprises an additional
C-terminal domain resulting in an engineered core TALE scaffold
sequentially comprising different sets of Repeat Variable Dipeptide
regions (RVDs) to change DNA binding specificity and target a
specific single double-stranded DNA target sequence of interest and
a C-terminal domain, onto which a selection of catalytic domains
can be attached to effect DNA processing.
[0055] In another embodiment, said engineered core TALE-scaffold
according to the present invention comprises additional N-terminus
and a C-terminal domains resulting in an engineered core TALE
scaffold sequentially comprising a N-terminal domain, different
sets of Repeat Variable Dipeptide regions (RVDs) to change DNA
binding specificity and target a specific single double-stranded
DNA target sequence of interest and a C-terminal domain, onto which
a selection of catalytic domains can be attached to effect DNA
processing. In another embodiment, said engineered core
TALE-scaffold according to the present invention comprises the
protein sequences selected from the group consisting of ST1 (SEQ ID
NO: 134) and ST2 (SEQ ID NO: 135). In another embodiment, said
engineered TALE-scaffold comprises a protein sequence having at
least 80%, more preferably 90%, again more preferably 95% amino
acid sequence identity with the protein sequences selected from the
group consisting of SEQ ID NO: 134 and SEQ ID NO: 135.
[0056] In another embodiment, said engineered core TALE-scaffold
according to the present invention comprises the protein sequences
selected from the group consisting of bT1-Avr (SEQ ID NO: 136),
bT2-Avr (SEQ ID NO: 137), bT1-Pth (SEQ ID NO: 138) and bT2-Pth (SEQ
ID NO: 139). In another embodiment, said engineered TALE-scaffold
comprises a protein sequence having at least 80%, more preferably
90%, again more preferably 95% amino acid sequence identity with
the protein sequences selected from the group consisting of SEQ ID
NO: 136 to SEQ ID NO: 139.
[0057] In a preferred embodiment according to the method of the
present invention, said additional N-terminus and C-terminal
domains of engineered core TALE scaffold are derived from natural
TALE. In a more preferred embodiment said additional N-terminus and
C-terminal domains of engineered core TALE scaffold are derived
from natural TALE like AvrBs3, PthXo1, AvrHah1, PthA, Tal1c as
non-limiting examples. In another more preferred embodiment, said
additional N-terminus and/or said C-terminal domains are truncated
forms of respective N-terminus and/or said C-terminal domains of
natural TALE like AvrBs3, PthXo1, AvrHah1, PthA, Tal1c as
non-limiting examples from which they are derived. In a more
preferred embodiment, said additional N-terminus and C-terminal
domains sequences of engineered core TALE scaffold are selected
from the group consisting of ST1 SEQ ID NO: 134 and ST2 SEQ ID NO:
135 as respectively exemplified in baseline protein scaffolds
bT1-Avr (SEQ ID NO: 136) or bT1-Pth (SEQ ID NO: 138) and bT2-Avr
(SEQ ID NO: 137) or bT2-Pth (SEQ ID NO: 139).
[0058] In another embodiment, each RVD of said core scaffold is
made of 30 to 42 amino acids, more preferably 33 or 34 wherein two
critical amino acids located at positions 12 and 13 mediates the
recognition of one nucleotide of said nucleic acid target sequence;
equivalent two critical amino acids can be located at positions
other than 12 and 13 specially in RVDs taller than 33 or 34 amino
acids long. Preferably, RVDs associated with recognition of the
different nucleotides are HD for recognizing C, NG for recognizing
T, NI for recognizing A, NN for recognizing G or A, NS for
recognizing A, C, G or T, HG for recognizing T, IG for recognizing
T, NK for recognizing G, HA for recognizing C, ND for recognizing
C, HI for recognizing C, HN for recognizing G, NA for recognizing
G, SN for recognizing G or A and YG for recognizing T, TL for
recognizing A, VT for recognizing A or G and SW for recognizing A.
More preferably, RVDs associated with recognition of the
nucleotides C, T, A, G/A and G respectively are selected from the
group consisting of NN or NK for recognizing G, HD for recognizing
C, NG for recognizing T and NI for recognizing A, TL for
recognizing A, VT for recognizing A or G and SW for recognizing A.
In another embodiment, RVDS associated with recognition of the
nucleotide C are selected from the group consisting of N* and RVDS
associated with recognition of the nucleotide T are selected from
the group consisting of N* and H*, where * denotes a gap in the
repeat sequence that corresponds to a lack of amino acid residue at
the second position of the RVD. In another embodiment, critical
amino acids 12 and 13 can be mutated towards other amino acid
residues in order to modulate their specificity towards nucleotides
A, T, C and G and in particular to enhance this specificity. By
other amino acid residues is intended any of the twenty natural
amino acid residues or unnatural amino acids derivatives.
[0059] In another embodiment, said core scaffold of the present
invention comprises between 8 and 30 RVDs. More preferably, said
core scaffold of the present invention comprises between 8 and 20
RVDs; again more preferably 15 RVDs.
[0060] In another embodiment, said core scaffold comprises an
additional single truncated RVD made of 20 amino acids located at
the C-terminus of said set of RVDs, i.e. an additional C-terminal
half-RVD. In this case, said core scaffold of the present invention
comprises between 8.5 and 30.5 RVDs, "0.5" referring to previously
mentioned half-RVD (or terminal RVD, or half-repeat). More
preferably, said core scaffold of the present invention comprises
between 8.5 and 20.5 RVDs, again more preferably, 15.5 RVDs. In a
preferred embodiment, said half-RVD is in a core scaffold context
which allows a lack of specificity of said half-RVD toward
nucleotides A, C, G, T. In a more preferred embodiment, said
half-RVD is absent.
[0061] In another embodiment, said core scaffold of the present
invention comprises RVDs of different origins. In a preferred
embodiment, said core scaffold comprises RVDs originating from
different naturally occurring TAL effectors. In another preferred
embodiment, internal structure of some RVDs of the core scaffold of
the present invention are constituted by structures or sequences
originated from different naturally occurring TAL effectors. In
another embodiment, said core scaffold of the present invention
comprises RVDs-like domains. RVDs-like domains have a sequence
different from naturally occurring RVDs but have the same function
and/or global structure within said core scaffold of the present
invention.
[0062] In another embodiment, said additional N-terminal domain of
said engineered core TALE scaffold is an enhancer domain. In
another embodiment, said enhancer domain is selected from the group
consisting of Puf RNA binding protein or Ankyrin super-family, as
non-limiting examples. In another embodiment, said enhancer domain
sequence is selected from the group consisting of protein domains
of SEQ ID NO: 4 and SEQ ID NO: 5, as non-limiting examples listed
in Table 1, a functional mutant, a variant or a derivative
thereof.
[0063] In another embodiment, said additional C-terminal domain of
said engineered core TALE scaffold is an enhancer domain. In
another embodiment, said enhancer domain is selected from the group
consisting of hydrolase/transferase of Pseudomonas Aeuriginosa
family, the polymerase domain from the Mycobacterium tuberculosis
Ligase D family, the initiation factor elF2 from Pyrococcus family,
the translation initiation factor Aif2 family, as non-limiting
examples. In another embodiment, said enhancer domain sequence is
selected from the group consisting of protein domains of SEQ ID NO:
6 to SEQ ID NO: 9, as non-limiting examples listed in Table 1, a
functional mutant, a variant or a derivative thereof.
TABLE-US-00001 TABLE 1 List of enhancer domains for engineered core
TALE scaffold. SEQ GENBANK/SWISS- ID PROT ID NAME NO FASTA SEQUENCE
gi|262368139|pdb| fem-3 4 >gi|262368139|pdb|3K5Q|A Chain A,
Crystal Structure Of Fbf-2FBE 3K5Q| COMPLEX
SNNVLPTWSLDSNGEMRSRLSLSEVLDSGDLMKFAVDKTGCQFLEKAVKGSLTSYQKFQLFEQV
IGRKDDFLKLSTNIFGNYLVQSVIGISLATNDDGYTKRQEKLKNFISSQMTDMCLDKFACRVIQ
SSLQNMDLSLACKLVQALPRDARLIAICVDQNANHVIQKVVAVIPLKNWEFIVDFVATPEHLRQ
ICSDKYGCRVVQTIIEKLTADSMNVDLTSAAQNLRERALQRLMTSVTNRCQELATNEYANYIIQ
HIVSNDDLAVYRECIIEKCLMRNLLSLSQEKFASHVVEKAFLHAPLELLAEMMDEIFDGYIPHP
DTGKDALDIMMFHQFGNYVVQCMLTICCDAVSGRRQTKEGGYDHAISFQDWLKKLHSRVTKERH
RLSRFSSGKKMIETLANLRSTHPIYELQ gi|308387836|pdb| aRep 5
>gi|308387836|pdb|3LTJ|A Chain A, Structure Of A New Family Of
3LTJ| Artificial Alpha Helicoidal Repeat Proteins (Alpha-Rep) Based
On Thermostable Heat-Like Repeats
MRGSHHHHHHTDPEKVEMYIKNLQDDSYYVRRAAAYALGKIGDERAVEPLIKALKDEDAWVRRA
AADALGQIGDERAVEPLIKALKDEDGWVRQSAAVALGQIGDERAVEPLIKALKDEDWFVRIAAA
FALGEIGDERAVEPLIKALKDEDGWVRQSAADALGEIGGERVRAAMEKLAETGTGFARKVAVNY
LETHKSLIS gi|109157579|pdb| Pseudomonas 6
>gi|109157579|pdb|2FAO|A Chain A, Crystal Structure Of 2FAO|
Aeruginosa Ligd Pseudomonas Aeruginosa Ligd Polymerase Domain
Polymerase
MGARKASAGASRAATAGVRISHPQRLIDPSIQASKLELAEFHARYADLLLRDLRERPVSLVRGP
Domain
DGIGGELFFQKHAARLKIPGIVQLDPALDPGHPPLLQIRSAEALVGAVQMGSIEFHTWNASLAN
LERPDRFVLDLDPDPALPWKRMLEATQLSLTLLDELGLRAFLKTSGGKGMHLLVPLERRHGWDE
VKDFAQAISQHLARLMPERFSAVSGPRNRVGKIFVDYLRNSRGASTVAAYSVRAREGLPVSVPV
FREELDSLQGANQWNLRSLPQRLDELAGDDPWADYAGTRQRISAAMRRQLGRG 2R9L_A GI:
Mycobacterium 7 Polymerase Domain From Mycobacterium Tuberculosis
Ligase D In 164519498 Tuberculosis Complex With Dna:Accession:
2R9L_A GI: 164519498 Ligase D >gi|164519498|pdb|2R9L|A Chain A,
Polymerase Domain From Mycobacterium Tuberculosis Ligase D In
Complex With Dna
GSHMGSASEQRVTLTNADKVLYPATGTTKSDIFDYYAGVAEVMLGHIAGRPATRKRWPNGVDQP
AFFEKQLALSAPPWLSRATVAHRSGTTTYPIIDSATGLAWIAQQAALEVHVPQWRFVAEPGSGE
LNPGPATRLVFDLDPGEGVMMAQLAEVARAVRDLLADIGLVTFPVTSGSKGLHLYTPLDEPVSS
RGATVLAKRVAQRLEQAMPALVTSTMTKSLRAGKVFVDWSQNSGSKTTIAPYSLRGRTHPTVAA
PRTWAELDDPALRQLSYDEVLTRIARDGDLLERLDADAPVADRLTRY 1KJZ_A Large Gamma
8 Structure Of The Large Gamma Subunit Of Initiation Factor Eif2
GI:20664108 Subunit Of From Pyrococcus Abyssi-G235d Mutant:1KJZ_A
GI:20664108, Initiation >gi|20664108|pdb|1KJZ|A Chain A,
Structure Of The Large Gamma Factor Eif2 Subunit Of Initiation
Factor Eif2 From Pyrococcus Abyssi-G235d From Pyrococcus Mutant
Abyssi
GEKRKSRQAEVNIGMVGHVDHGKTTLTKALTGVWTDTHSEELRRGITIKIGFADAEIRRCPNCG
RYSTSPVCPYCGHETEFVRRVSFIDAPGHEALMTTMLAGASLMDGAILVIAANEPCPRPQTREH
LMALQIIGQKNIIIAQNKIELVDKEKALENYRQIKEFIEGTVAENAPIIPISALHGANIDVLVK
AIEDFIPTPKRDPNKPPKMLVLRSFDVNKPGTPPEKLVGGVLDGSIVQGKLKVGDEIEIRPGVP
YEEHGRIKYEPITTEIVSLQAGGQFVEEAYPGGLVGVGTKLDPYLTKGDLMAGNVVGKPGKLPP
VWDSLRLEVHLLERVVGTEQELKVEPIKRKEVLLLNVGTARTMGLVTGLGKDEIEVKLQIPVCA
EPGDRVAISRQIGSRWRLIGYGIIKE 2D74_A Translation 9 Crystal Structure
Of Translation Initiation Factor Aif2betagamma GI:112490420
Initiation Heterodimer:2D74_A GI:112490420, Factor
>gi|112490420|pdb|2D74|A Chain A, Crystal Structure Of
Aif2betagamma Translation Initiation Factor Aif2betagamma
Heterodimer
MGEKRKTRQAEVNIGMVGHVDHGKTTLTKALTGVWTDTHSEELRRGITIKIGFADAEIRRCSNC
GRYSTSPICPYCGHETEFIRRVSFIDSPGHEALMTTMLAGASLMDGAILVIAANEPCPRPQTRE
HLMALQIIGQKNIIIAQNKIELVDKEKALENYRQIKEFIKGTVAENAPIIPISALHGANIDVLV
KAIEEFIPTPKRDSNKPPKMLVLRSFDVNKPGTPPEKLVGGVLDGSIVQGKLKVGDEIEIRPGV
PYEEHGRIKYEPITTEIVSLQAGGQFVEEAYPGGLVGIGTKLDPYLTKGDLMAGNVVGKPGKLP
PVWTDLRLEVHLLERVVGTEQELNVEPIKRKEVLLLNVGTARTMGLVTALGKDEIELKLQIPVC
AEPGERVAISRQIGSRWRLIGYGIIKELEHHHHHH
[0064] In another preferred embodiment according to the method of
the present invention, the catalytic domain that is capable of
processing DNA nearby the single double-stranded DNA target
sequence of interest, when fused to said engineered core TALE
scaffold according to the method of the present invention, is fused
to the N-terminus part of said core TALE scaffold. In another
preferred embodiment, said catalytic domain is fused to the
C-terminus part of said core TALE scaffold. In another preferred
embodiment two catalytic domains are fused to both N-terminus part
of said core TALE scaffold and C-terminus part of said core TALE
scaffold. In a more preferred embodiment, said catalytic domain has
an enzymatic activity selected from the group consisting of
nuclease activity, polymerase activity, kinase activity,
phosphatase activity, methylase activity, topoisomerase activity,
integrase activity, transposase activity or ligase activity. In
another preferred embodiment, the catalytic domain fused to the
core TALE scaffold of the present invention can be a transcription
activator or repressor (i.e. a transcription regulator), or a
protein that interacts with or modifies other proteins such as
histones. Non-limiting examples of DNA processing activities of
said compact TALEN of the present invention include, for example,
creating or modifying epigenetic regulatory elements, making
site-specific insertions, deletions, or repairs in DNA, controlling
gene expression, and modifying chromatin structure.
[0065] In another more preferred embodiment, said catalytic domain
has an endonuclease activity. In another more preferred embodiment,
said catalytic domain has cleavage activity on said double-stranded
DNA according to the method of the present invention. In another
more preferred embodiment, said catalytic domain has a nickase
activity on said double-stranded DNA according to the method of the
present invention. In another more preferred embodiment, said
catalytic domain is selected from the group consisting of proteins
MmeI, Colicin-E7 (CEA7_ECOLX), Colicin-E9, APFL, EndA, Endo I
(END1_ECOLI), Human Endo G (NUCG_HUMAN), Bovine Endo G
(NUCG_BOVIN), R.HinP1I, I-BasI, I-BmoI, I-HmuI, I-TevI, I-TevII,
I-TevIII, I-TwoI, R.MspI, R.MvaI, NucA, NucM, Vvn, Vvn_CLS,
Staphylococcal nuclease (NUC_STAAU), Staphylococcal nuclease
(NUC_STAHY), Micrococcal nuclease (NUC_SHIFL), Endonuclease yncB,
Endodeoxyribonuclease I (ENRN_BPT7), Metnase, Nb.BsrDI, BsrDI A,
Nt.BspD6I (R.BspD6I large subunit), ss.BspD6I (R.BspD6I small
subunit), R.PleI, MlyI, AlwI, Mva1269I, BsrI, BsmI, Nb.BtsCI,
Nt.BtsCI, R1.BtsI, R2.BtsI, BbvCI subunit 1, BbvCI subunit 2,
Bpu10I alpha subunit, Bpu10I beta subunit, BmrI, BfiI, I-CreI,
hExol (EXO1_HUMAN), Yeast Exol (EXO1_YEAST), E. coli Exol, Human
TREX2, Mouse TREX1, Human TREX1, Bovine TREX1, Rat TREX1, Human
DNA2, Yeast DNA2 (DNA2_YEAST) and VP16, as listed in Table 2 (SEQ
ID NO: 10 to SEQ ID NO: 66 and SEQ ID NO: 1, 366 & 367), a
functional mutant, a variant or a derivative thereof. In another
preferred embodiment according to the method of the present
invention, said catalytic domain is I-TevI (SEQ ID NO: 20), a
functional mutant, a variant or a derivative thereof. In another
preferred embodiment, catalytic domain I-TevI (SEQ ID NO: 20), a
functional mutant, a variant or a derivative thereof is fused to
the N-terminal domain of said core TALE scaffold according to the
method of the present invention. In another preferred embodiment,
said compact TALEN according to the method of the present invention
comprises a protein sequence having at least 80%, more preferably
90%, again more preferably 95% amino acid sequence identity with
the protein sequences selected from the group of SEQ ID NO:
420-432.
[0066] In another preferred embodiment, said catalytic domain is
ColE7 (SEQ ID NO: 11), a functional mutant, a variant or a
derivative thereof. In another preferred embodiment, catalytic
domain ColE7 (SEQ ID NO: 11), a functional mutant, a variant or a
derivative thereof is fused to the N-terminal domain of said core
TALE scaffold according to the method of the present invention. In
another preferred embodiment, catalytic domain ColE7 (SEQ ID NO:
11), a functional mutant, a variant or a derivative thereof is
fused to the C-terminal domain of said core TALE scaffold according
to the method of the present invention. In another preferred
embodiment, said compact TALEN according to the method of the
present invention comprises a protein sequence having at least 80%,
more preferably 90%, again more preferably 95% amino acid sequence
identity with the protein sequences selected from the group of SEQ
ID NO: 435-438.
[0067] In another preferred embodiment, said catalytic domain is
NucA (SEQ ID NO: 26), a functional mutant, a variant or a
derivative thereof. In another preferred embodiment, catalytic
domain NucA (SEQ ID NO: 26), a functional mutant, a variant or a
derivative thereof is fused to the N-terminal domain of said core
TALE scaffold according to the method of the present invention. In
another preferred embodiment, catalytic domain NucA (SEQ ID NO:
26), a functional mutant, a variant or a derivative thereof is
fused to the C-terminal domain of said core TALE scaffold according
to the method of the present invention. In another preferred
embodiment, said compact TALEN according to the method of the
present invention comprises a protein sequence having at least 80%,
more preferably 90%, again more preferably 95% amino acid sequence
identity with the protein sequences selected from the group of SEQ
ID NO: 433-434.
[0068] In another preferred embodiment, said catalytic domain is
I-CreI (SEQ ID NO: 1), a functional mutant, a variant or a
derivative thereof. In another preferred embodiment, catalytic
domain I-CreI (SEQ ID NO: 1), a functional mutant, a variant or a
derivative thereof is fused to the N-terminal domain of said core
TALE scaffold according to the method of the present invention. In
another preferred embodiment, catalytic domain I-CreI (SEQ ID NO:
1), a functional mutant, a variant or a derivative thereof is fused
to the C-terminal domain of said core TALE scaffold according to
the method of the present invention. In another preferred
embodiment, said compact TALEN according to the method of the
present invention comprises a protein sequence having at least 80%,
more preferably 90%, again more preferably 95% amino acid sequence
identity with the protein sequences selected from the group of SEQ
ID NO: 439-441 and SEQ ID NO: 444-446.
[0069] In another embodiment, said catalytic domain is a
restriction enzyme such as MmeI, R-HinPII, R.MspI, R.MvaI,
Nb.BsrDI, BsrDI A, Nt.BspD6I, ss.BspD6I, R.PleI, MlyI and AlwI as
non-limiting examples listed in table 2. In another more preferred
embodiment, said catalytic domain has an exonuclease activity.
[0070] In another more preferred embodiment, any combinations of
two catalytic domains selected from the group consisting of
proteins MmeI, Colicin-E7 (CEA7_ECOLX), Colicin-E9, APFL, EndA,
Endo I (END1_ECOLI), Human Endo G (NUCG_HUMAN), Bovine Endo G
(NUCG_BOVIN), R.HinP1I, I-BasI, I-BmoI, I-HmuI, I-TevI, I-TevII,
I-TevIII, I-TwoI, R.MspI, R.MvaI, NucA, NucM, Vvn, Vvn_CLS,
Staphylococcal nuclease (NUC_STAAU), Staphylococcal nuclease
(NUC_STAHY), Micrococcal nuclease (NUC_SHIFL), Endonuclease yncB,
Endodeoxyribonuclease I (ENRN_BPT7), Metnase, Nb.BsrDI, BsrDI A,
Nt.BspD6I (R.BspD6I large subunit), ss.BspD6I (R.BspD6I small
subunit), R.PleI, MlyI, AlwI, Mva1269I, BsrI, BsmI, Nb.BtsCI,
Nt.BtsCI, R1.BtsI, R2.BtsI, BbvCI subunit 1, BbvCI subunit 2,
Bpu10I alpha subunit, Bpu10I beta subunit, BmrI, BfiI, I-CreI,
hExol (EXO1_HUMAN), Yeast Exol (EXO1_YEAST), E. coli Exol, Human
TREX2, Mouse TREX1, Human TREX1, Bovine TREX1, Rat TREX1, Human
DNA2, Yeast DNA2 (DNA2_YEAST) and VP16, as listed in Table 2 (SEQ
ID NO: 10 to SEQ ID NO: 66 and SEQ ID NO: 1, 366 & 367), a
functional mutant, a variant or a derivative of these protein
domains thereof, can be fused to both N-terminus part and
C-terminus part of said core TALE scaffold, respectively. For
example, I-HmuI catalytic domain can be fused to the N-terminus
part of said core TALE scaffold and ColE7 catalytic domain can be
fused to the C-terminus part of said core TALE scaffold. In another
example, I-TevI catalytic domain can be fused to the N-terminus
part of said core TALE scaffold and ColE7 catalytic domain can be
fused to the C-terminus part of said core TALE scaffold. In another
embodiment, according to the method of the present invention, said
unique compact TALEN monomer comprises a combination of two
catalytic domains respectively fused to the C-terminus part and to
the N-terminus part of said core TALE scaffold selected from the
group consisting of: [0071] (i) A Nuc A domain (SEQ ID NO: 26) in
N-terminus and a Nuc A domain (SEQ ID NO: 26) in C-terminus; [0072]
(ii) A ColE7 domain (SEQ ID NO: 11) in N-terminus and a ColE7
domain (SEQ ID NO: 11) in C-terminus; [0073] (iii) A TevI domain
(SEQ ID NO: 20) in N-terminus and a ColE7 domain (SEQ ID NO: 11) in
C-terminus; [0074] (iv) A TevI domain (SEQ ID NO: 20) in N-terminus
and a NucA domain (SEQ ID NO: 26) in C-terminus; [0075] (v) A ColE7
domain (SEQ ID NO: 11) in N-terminus and a NucA domain (SEQ ID NO:
26) in C-terminus; [0076] (vi) A NucA domain (SEQ ID NO: 26) in
N-terminus and a ColE7 domain (SEQ ID NO: 11) in C-terminus.
[0077] In another preferred embodiment, said compact TALEN
according to the method of the present invention comprises a
protein sequence having at least 80%, more preferably 90%, again
more preferably 95% amino acid sequence identity with the protein
sequences selected from the group consisting of SEQ ID NO: 448 and
450.
[0078] In another preferred embodiment, said compact TALEN
according to the method of the present invention comprises a
combination of two catalytic domains respectively fused to the
C-terminus part and to the N-terminus part of said core TALE
scaffold selected from the group consisting of: [0079] (i) A TevI
domain (SEQ ID NO: 20) in N-terminus and a FokI domain (SEQ ID NO:
368) in C-terminus; [0080] (ii) A TevI domain (SEQ ID NO: 20) in
N-terminus and a TevI domain (SEQ ID NO: 20) in C-terminus; [0081]
(iii) A scTrex2 domain (SEQ ID NO: 451) in N-terminus and a FokI
domain (SEQ ID NO: 368) in C-terminus.
[0082] In another preferred embodiment, said compact TALEN
according to the method of the present invention comprises a
protein sequence having at least 80%, more preferably 90%, again
more preferably 95% amino acid sequence identity with the protein
sequences selected from the group consisting of SEQ ID NO: 447-450
and SEQ ID NO: 452.
[0083] In the scope of the present invention, it can be envisioned
to insert said catalytic domain between two parts of the engineered
core TALE scaffold according to the invention, each part comprising
one set of RVDs. In this last case, the number of RVDs for each
part of the engineered core TALE scaffold can be the same or not.
In other words, it can be envisioned to split said core TALE
scaffold of the present invention to insert one catalytic domain
between the resulting two parts of said engineered core TALE
scaffold. In another preferred embodiment, said compact TALEN
according to the method of the present invention comprises a
protein sequence having at least 80%, more preferably 90%, again
more preferably 95% amino acid sequence identity with the protein
sequences selected from the group consisting of SEQ ID NO:
453-455.
TABLE-US-00002 TABLE 2 List of catalytic/enhancer domains for
compact TALENs or enhanced compact TALENs. GENBANK/ SWISS- SEQ ID
PROT ID NAME NO FASTA SEQUENCE ACC85607.1 MmeI 10
>gi|186469979|gb|ACC85607.1| MmeI [Methylophilus methylotrophus]
MALSWNEIRRKAIEFSKRWEDASDENSQAKPFLIDFFEVFGITNKRVATFEHAVKKFAKAHKEQSRGFVD
LFWPGILLIEMKSRGKDLDKAYDQALDYFSGIAERDLPRYVLVCDFQRFRLTDLITKESVEFLLKDLYQN
VRSFGFIAGYQTQVIKPQDPINIKAAERMGKLHDTLKLVGYEGHALELYLVRLLFCLFAEDTTIFEKSLF
QEYIETKTLEDGSDLAHHINTLFYVLNTPEQKRLKNLDEHLAAFPYINGKLFEEPLPPAQFDKAMREALL
DLCSLDWSRISPAIFGSLFQSIMDAKKRRNLGAHYTSEANILKLIKPLFLDELWVEFEKVKNNKNKLLAF
HKKLRGLTFFDPACGCGNFLVITYRELRLLEIEVIRGLHRGGQQVLDIEHLIQINVDQFFGIEIEEFPAQ
IAQVALWLTDHQMNMKISDEFGNYFARIPLKSTPHILNANALQIDWNDVLEAKKCCFILGNPPFVGKSKQ
TPGQKADLLSVFGNLKSASDLDLVAAWYPKAAHYIQTNANIRCAFVSTNSITQGEQVSLLWPLLLSLGIK
INFAHRTFSWTNEASGVAAVHCVIIGFGLKDSDEKIIYEYESINGEPLAIKAKNINPYLRDGVDVIACKR
QQPISKLPSMRYGNKPTDDGNFLFTDEEKNQFITNEPSSEKYFRRFVGGDEFINNTSRWCLWLDGADISE
IRAMPLVLARIKKVQEFRLKSSAKPTRQSASTPMKFFYISQPDTDYLLIPETSSENRQFIPIGFVDRNVI
SSNATYHIPSAEPLIFGLLSSTMHNCWMRNVGGRLESRYRYSASLVYNTFPWIQPNEKQSKAIEEAAFAI
LKARSNYPNESLAGLYDPKTMPSELLKAHQKLDKAVDSVYGFKGPNTEIARIAFLFETYQKMTSLLPPEK
EIKKSKGKN Q47112.2 Colicin-E7 11
>gi|12644448|sp|Q47112.2|CEA7_ECOLX RecName: Full = Colicin-E7
(CEA7_ECOLX)
MSGGDGRGHNSGAHNTGGNINGGPTGLGGRGGASDGSGWSSENNPWGGGSGSGVHWGGGSGHGNGGGNSN
SGGGSNSSVAAPMAFGFPALAAPGAGTLGISVSGEALSAAIADIFAALKGPFKFSAWGIALYGILPSEIA
KDDPNMMSKIVTSLPAETVTNVQVSTLPLDQATVSVTKRVTDVVKDTRQHIAVVAGVPMSVPVVNAKPTR
TPGVFHASFPGVPSLTVSTVKGLPVSTTLPRGITEDKGRTAVPAGFTFGGGSHEAVIRFPKESGQKPVYV
SVTDVLTPAQVKQRQDEEKRLQQEWNDAHPVEVAERNYEQARAELNQANKDVARNQERQAKAVQVYNSRK
SELDAANKTLADAKAEIKQFERFAREPMAAGHRMWQMAGLKAQRAQTDVNNKKAAFDAAAKEKSDADVAL
SSALERRKQKENKEKDAKAKLDKESKRNKPGKATGKGKPVNNKWLNNAGKDLGSPVPDRIANKLRDKEFK
SFDDFRKKFWEEVSKDPELSKQFSRNNNDRMKVGKAPKTRTQDVSGKRTSFELHHEKPISQNGGVYDMDN
ISVVTPKRHIDIHRGK CAA38134.1 EndA 12 >gi|47374|emb|CAA38134.1|
EndA [Streptococcus pneumoniae]
MNKKTROTLIGLLVLLLLSTGSYYIKQMPSAPNSPKTNLSQKKQASEAPSQALAESVLTDAVKSQIKGSL
EWNGSGAFIVNGNKTNLDAKVSSKPYADNKTKTVGKETVPTVANALLSKATRQYKNRKETGNGSTSWTPP
GWHQVKNLKGSYTHAVDRGHLLGYALIGGLDGFDASTSNPKNIAVQTAWANQAQAEYSTGQNYYESKVRK
ALDQNKRVRYRVTLYYASNEDLVPSASQIEAKSSDGELEFNVLVPNVQKGLQLDYRTGEVTVTQ
P25736.1 Endo I 13 >gi|119325|sp|P25736.1|END1_ECOLI RecName:
Full = Endonuclease-1; (END1_ECOLI) AltName: Full = Endonuclease I;
Short = Endo I; Flags: Precursor
MYRYLSIAAVVLSAAFSGPALAEGINSFSQAKAAAVKVHADAPGTFYCGCKINWQGKKGVVDLQSCGYQV
RKNENRASRVEWEHVVPAWQFGHQRQCWQDGGRKNCAKDPVYRKMESDMHNLQPSVGEVNGDRGNFMYSQ
WNGGEGQYGQCAMKVDFKEKAAEPPARARGAIARTYFYMRDQYNLTLSRQQTQLFNAWNKMYPVTDWECE
RDERIAKVQGNHNPYVQRACQARKS Q14249.4 Human Endo G 14
>gi|317373579|sp|Q14249.4|NUCG_HUMAN RecName: Full =
Endonuclease G, (NUCG_HUMAN) mitochondrial; Short = Endo G; Flags:
Precursor
MRALRAGLTLASGAGLGAVVEGWRARREDARAAPGLLGRLPVLPVAAAAELPPVPGGPRGPGELAKYGLP
GLAQLKSRESYVLCYDPRTRGALWVVEQLRPERLRGDGDRRECDFREDDSVHAYHRATNADYRGSGFDRG
HLAAAANHRWSQKAMDDTFYLSNVAPQVPHLNQNAWNNLEKYSRSLTRSYQNVYVCTGPLFLPRTEADGK
SYVKYQVIGKNHVAVPTHFFKVLILEAAGGQIELRTYVMPNAPVDEAIPLERFLVPIESIERASGLLFVP
NILARAGSLKAITAGSK P38447.1 Bovine Endo G 15
>gi|585596|sp|P38447.1|NUCG_BOVIN RecName: Full = Endonuclease
G, (NUCG_BOVIN) mitochondrial; Short = Endo G; Flags: Precursor
MQLLRAGLTLALGAGLGAAAESWWRQRADARATPGLLSRLPVLPVAAAAGLPAVPGAPAGGGPGELAKYG
LPGVAQLKSRASYVLCYDPRTRGALWVVEQLRPEGLRGDGNRSSCDFHEDDSVHAYHRATNADYRGSGFD
RGHLAAAANHRWSQKAMDDTFYLSNVAPQVPHLNQNAWNNLEKYSRSLTRTYQNVYVCTGPLFLPRTEAD
GKSYVKYQVIGKNHVAVPTHFFKVLILEAAGGQIELRSYVMPNAPVDEAIPLEHFLVPIESIERASGLLF
VPNILARAGSLKAITAGSK AAW33811.1 R.HinP1I 16
>gi|57116674|gb|AAW33811.1| R.HinP1I restriction endonuclease
[Haemophilus influenzae]
MNLVELGSKTAKDGFKNEKDIADRFENWKENSEAQDWLVTMGHNLDEIKSVKAVVLSGYKSDINVQVLVF
YKDALDIHNIQVKLVSNKRGFNQIDKHWLAHYQEMWKFDDNLLRILRHFTGELPPYHSNTKDKARMFMTE
FSQEEQNIVLNWLEKNRVLVLTDILRGRGDFAAEWVLVAQKVSNNARWILRNINEVLQHYGSGDISLSPR
GSINFGRVTIQRKGGDNGRETANMLQFKIDPTELFDI AAO93095.1 I-BasI 17
>gi|29838473|gb|AAO93095.1| I-BasI [Bacillus phage Bastille]
MFQEEWKDVTGFEDYYEVSNKGRVASKRTGVIMAQYKINSGYLCIKFTVNKKRTSHLVHRLVAREFCEGY
SPELDVNHKDTDRMNNNYDNLEWLTRADNLKDVRERGKLNTHTAREALAKVSKKAVDVYTKDGSEYIATY
PSATEAAEALGVQGAKISTVCHGKRQHTGGYHFKFNSSVDPNRSVSKK AAK09365.1 I-BmoI
18 >gi|12958590|gb|AAK09365.1|AF321518_2 intron encoded I-BmoI
[Bacillus mojavensis]
MKSGVYKITNKNTGKFYIGSSEDCESRLKVHFRNLKNNRHINRYLNNSFNKHGEQVFIGEVIHILPIEEA
IAKEQWYIDNFYEEMYNISKSAYHGGDLTSYHPDKRNIILKRADSLKKVYLKMTSEEKAKRWQCVQGENN
PMFGRKHTETTKLKISNHNKLYYSTHKNPFKGKKHSEESKTKLSEYASQRVGEKNPFYGKTHSDEFKTYM
SKKFKGRKPKNSRPVIIDGTEYESATEASRQLNVVPATILHRIKSKNEKYSGYFYK P34081.1
I-HmuI 19 >gi|465641|sp|P34081.1|HMUI_BPSP1 RecName: Full = DNA
endonuclease I- HmuI; AltName: Full = HNH homing endonuclease
I-HmuI
MEWKDIKGYEGHYQVSNTGEVYSIKSGKTLKHQIPKDGYHRIGLFKGGKGKTFQVHRLVAIHFCEGYEEG
LVVDHKDGNKDNNLSTNLRWVTQKINVENQMSRGTLNVSKAQQIAKIKNQKPIIVISPDGIEKEYPSTKC
ACEELGLTRGKVTDVLKGHRIHHKGYTFRYKLNG P13299.2 I-TevI 20
>gi|6094464|sp|P13299.2|TEV1_BPT4 RecName: Full =
Intron-associated endonuclease 1; AltName: Full = I-TevI; AltName:
Full = IRF protein
MKSGIYQIKNTLNNKVYVGSAKDFEKRWKRHFKDLEKGCHSSIKLQRSFNKHGNVFECSILEEIPYEKDL
IIERENFWIKELNSKINGYNIADATFGDTCSTHPLKEEIIKKRSETVKAKMLKLGPDGRKALYSKPGSKN
GRWNPETHKFCKCGVRIQTSAYTCSKCRNRSGENNSFFNHKESDITKSKISEKMKGKKPSNIKKISCDGV
IFDCAADAARHFKISSGLVTYRVKSDKWNWFYINA P07072.2 I-TevII 21
>gi|20141823|sp|P07072.2|TEV2_BPT4 RecName: Full =
Intron-associated endonuclease 2; AltName: Full = I-TevII
MKWKLRKSLKIANSVAFTYMVRFPDKSFYIGFKKFKTIYGKDTNWKEYNSSSKLVKEKLKDYKAKWIILQ
VFDSYESALKHEEMLIRKYFNNEFILNKSIGGYKFNKYPDSEEHKQKLSNAHKGKILSLKHKDKIREKLI
EHYKNNSRSEAHVKNNIGSRTAKKTVSIALKSGNKFRSFKSAAKFLKCSEEQVSNHPNVIDIKITIHPVP
EYVKINDNIYKSFVDAAKDLKLHPSRIKDLCLDDNYPNYIVSYKRVEK Q38419.1 I-TevIII
22 >gi|11387192|sp|Q38419.1|TEV3_BPR03 RecName: Full =
Intron-associated endonuclease 3; AltName: Full = I-TevIII
MNYRKIWIDANGPIPKDSDGRTDEIHHKDGNRENNDLDNLMCLSIQEHYDIHLAQKDYQACHAIKLRMKY
SPEEISELASKAAKSREIQIFNIPEVRAKNIASIKSKIENGTFHLLDGEIQRKSNLNRVALGIHNFQQAE
HIAKVKERNIAAIKEGTHVFCGGKMQSETQSKRVNDGSHHFLSEDHKKRTSAKTLEMVKNGTHPAQKEIT
CDFCGHIGKGPGFYLKHNDRCKLNPNRIQLNCPYCDKKDLSPSTYKRWHGDNCKARFND
AAM00817.1 I-TwoI 23 >gi|19881200|gb|AAM00817.1|AF485080_2 HNH
endonuclease I-TwoI [Staphylococcus phage Twort]
MEELWKEIPGFNSYMISNKGQVYSRKANKILALRTDKNGYKRISIFNNEGKRILLGVHKLVLLGFKGINT
EKPIPHHKNNIKDDNRLENLEWVTVSENTKHAYDIGALKSPRRVTCTLYYKGEPLSCYDSLFDLAKALKV
SRSVIESPRNGLVLSTFEVKREPTIQGLPLNKEIFEHSLIKGLGNPPLKVYNEDETYYFLTLMDISKYFN
ESYSKVQRGYYKGKWKSYIIEHIDFYEYYKQTH P11405.1 R.MspI 24
>gi|135239|sp|P11405.1|T2M1_MORSP RecName: Full = Type-2
restriction enzyme MspI; Short = R.MspI; FatName: Full =
Endonuclease MspI; AltName: Full = Type II restriction enzyme MspI
MRTELLSKLYDDFGIDQLPHTQHGVTSDRLGKLYEKYILDIFKDIESLKKYNTNAFPQEKDISSKLLKAL
NLDLDNIIDVSSSDTDLGRTIAGGSPKTDATIRFTFHNQSSRLVPLNIKHSSKKKVSIAEYDVETICTGV
GISDGELKELIRKHQNDQSAKLFTPVQKQRLTELLEPYRERFIRWCVTLRAEKSEGNILHPDLLIRFQVI
DREYVDVTIKNIDDYVSDRIAEGSKARKPGFGTGLNWTYASGSKAKKMQFKG R.MvaI R.MvaI
25 >gi|119392963|gb|AAM03024.2|AF472612_1 R.MvaI [Kocuria
varians]
MSEYLNLLKEAIQNVVDGGWHETKRKGNTGIGKTFEDLLEKEEDNLDAPDFHDIEIKTHETAAKSLLTLF
TKSPTNPRGANTMLRNRYGKKDEYGNNILHQTVSGNRKTNSNSYNYDFKIDIDWESQVVRLEVFDKQDIM
IDNSVYWSFDSLQNQLDKKLKYIAVISAESKIENEKKYYKYNSANLFTDLTVQSLCRGIENGDIKVDIRI
GAYHSGKKKGKTHDHGTAFRINMEKLLEYGEVKVIV CAA45962.1 NucA 26
gi|39041|emb|CAA45962.1| NucA [Nostoc sp. PCC 7120]
MGICGKLGVAALVALIVGCSPVQSQVPPLTELSPSISVELLLGNPSGATPTKLTPDNYLMVXNQYALSYN
NSKGTANWVAWQLNSSWLGNAERQDNFRPDKTLPAGWVRVTPSMYSGSGYDRGHIAPSADRTKTTEDNAA
TFLMTNMMPQTPDNNRNTWGNLEDYCRELVSQGKELYIVAGPNGSLGKPLKGKVTVPKSTWKIVVVLDSP
GSGLEGITANTRVIAVNIPNDPELNNDWRAYKVSVDELESLTGYDFLSNVSPNIQTSIESKVDN
P37994.2 NucM 27 gi|313104150|sp|P37994.2|NUCM_DICD3 RecName: Full
Nuclease nucM; Flags: Precursor
MLRNLVIFAVLGAGLTTLAAAGQDINNFTQAKAAAAKIHQDAPGTFYCGCKINWQGKKGTPDLASCGYQV
RKDANRASRIEWEHVVPAWQFGHQRQCWQDGGRKNCTKDDVYRQIETDLHNLQPAIGEVNGDRGNFMYSQ
WNGGERQYGQCEMKIDFKSQLAEPPERARGAIARTYFYMRDRYNLNLSRQQTQLFDAWNKQYPATTWECT
REKRIAAVQGNHNPYVQQACQP AAF19759.1 Vvn 28
>gi|6635279|gb|AAF19759.1|AF063303_1 nuclease precursor Vvn
[Vibrio vulnificus]
MKRLFIFIASFTAFAIQAAPPSSFSAAKQQAVKIYQDHPISFYCGCDIEWQGKKGIPNLETCGYQVRKQQ
TRASRIEWEHVVPAWQFGHHRQCWQKGGRKNCSKNDQQFRLMEADLHNLTPAIGEVNGDRSNFNFSQWNG
VDGVSYGRCEMQVNFKQRKVMPQTELRGSIARTYLYMSQEYGFQLSKQQQQLMQAWNKSYPVDEWECTRD
DRIAKIQGNHNPFVQQSCQTQ AAF19759.1 Vvn_CLS 29 >Vvn_CLS (variant of
AAF19759.1) (reference)
MASGAPPSSFSAAKQQAVKIYQDHPISFYCGCDIEWQGKKGIPNLETCGYQVRKQQTRASRIEWEHVVPA
WQFGHHRQCWQKGGRKNCSKNDQQFRLMEADLHNLTPAIGEVNGDRSNFNFSQWNGVDGVSYGRCEMQVN
FKQRKVMPPDRARGSIARTYLYMSQEYGFQLSKQQQQLMQAWNKSYPVDEWECTRDDRIAKIQGNHNPFV
QQSCQTQGSSAD P00644.1 Staphylococcal 30
>gi|128852|sp|P00644.1|NUC_STAAU RecName: Full = Thermonuclease;
nuclease Short = TNase; AltName: Full = Micrococcal nuclease;
AltName: (NUC_STAAU) Full = Staphylococcal nuclease; Contains:
RecName: Full = Nuclease B; Contains: RecName: Full = Nuclease A;
Flags: Precursor
MLVMTEYLLSAGICMAIVSILLIGMAISNVSKGQYAKREFFFATSCLVLTLVVVSSLSSSANASQTDNGV
NRSGSEDPTVYSATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETKHPKKGVEKYGPEA
SAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALVRQGLAKVAYVYKPNNTHEQHLRKS
EAQAKKEKLNIWSEDNADSGQ P43270.1 Staphylococcal 31
>gi|1171859|sp|P43270.1|NUC_STAHY RecName: Full =
Thermonuclease; nuclease Short = TNase; AltName: Full = Micrococcal
nuclease; AltName: (NUC_STAHY) Full = Staphylococcal nuclease;
Flags: Precursor
MKKITTGLIIVVAAIIVLSIQFMTESGPFKSAGLSNANEQTYKVIRVIDGDTIIVDKDGKQQNLRMIGVD
TPETVKPNTPVQPYGREASDETKRHLTNQKVALEYDKQEKDRYGRTLAYVWLGKEMENEKLAKEGLARAK
FYRPNYKYQERIEQAQKQAQKLKKNIWSN P29769.1 Micrococcal 32
>gi|266681|sp|P29769.1|NUC_SHIFL RecName: Full = Micrococcal
nuclease; nuclease Flags: Precursor (NUC_SHIFL)
MKSALAALRAVAAAVVLIVSVPAWADFRGEVVRILDGDTIDVLVNRQTIRVRLADIDAPESGQAFGSRAR
QRLADLTFRQEVQVTEKEVDRYGRTLGVVYAPLQYPGGQTQLTNINAIMVQEGMAWAYRYYGKPTDAQMY
EYEKEARRQRLGLWSDPNAQEPWKWRRASKNATN P94492.1 Endonuclease 33
gi|81345826|sp||YNCB_BACSU RecName: Full = Endonuclease yncB;
Flags: yncB Precursor
MKKILISMIAIVLSITLAACGSNHAAKNHSDSNGTEQVSQDTHSNEYNQTEQKAGTPHSKNQKKLVNVTL
DRAIDGDTIKVIYNGKKDTVRYLLVDTPETKKPNSCVQPYGEDASKRNKELVNSGKLQLEFDKGDRRDKY
GALLAYVYVDGKSVQETLLKEGLARVAYVYEPNTKYIDQFRLDEQEAKSDKLSIWSKSGYVTNRGFNGCV
K P00641.1 Endodeoxy- 34 >gi|119370|sp|P00641.1|ENRN_BPT7
RecName: Full = Endodeoxyribonuclease ribonu- 1; AltName: Full =
Endodeoxyribonuclease I; Short = Endonuclease clease I
MAGYGAKGIRKVGAFRSGLEDKVSKQLESKGIKFEYEEWKVPYVIPASNHTYTPDFLLPNGIF-
VETKGLW (ENRN_BPT7)
ESDDRKKHLLIREQHPELDIRIVFSSSRTKLYKGSPTSYGEFCEKHGIKFADKLIPAEWIKEPKKEVPFD
RLKRKGGKK Q53H47.1 Metnase 35
>gi|74740552|sp|Q53H47.1|SETMR_HUMAN RecName: Full =
Histone-lysine N- methyltransferase SETMAR; AltName: Full = SET
domain and mariner transposase fusion gene-containing protein;
Short = HsMar1; Short = Metnase; Includes: RecName: Full =
Histone-lysine N-methyl- transferase; Includes: RecName: Full =
Mariner transposase Hsmar1
MAEFKEKPEAPTEQLDVACGQENLPVGAWPPGAAPAPFQYTPDHVVGPGADIDPTQITFPGCICVKTPCL
PGTCSCLRHGENYDDNSCLRDIGSGGKYAEPVFECNVLCRCSDHCRNAVVQKGLQFHFQVFKTHKKGWGL
RTLEFIPKGRFVCEYAGEVLGFSEVQRRIHLQTKSDSNYIIAIREHVYNGQVMETFVDPTYIGNIGRFLN
HSCEPNLLMIPVRIDSMVPKLALFAAKDIVPEEELSYDYSGRYLNLTVSEDKERLDHGKLRKPCYCGAKS
CTAFLPFDSSLYCPVEKSNISCGNEKEPSMCGSAPSVFPSCKRLTLETMKMMLDKKQIRAIFLFEFKMGR
KAAETTRNINNAFGPGTANERTVQWWFKKFCKGDESLEDEERSGRPSEVDNDQLRAIIEADPLTTTREVA
EELNVNHSTVVRHLKQIGKVKKLDKWVPHELTENQKNRRFEVSSSLILRNHNEPFLDRIVTCDEKWILYD
NRRRSAQWLDQEEAPKHFPKPILHPKKVMVTIWWSAAGLIHYSFLNPGETITSEKYAQEIDEMNQKLQRL
QLALVNRKGPILLHDNARPHVAQPTLQKLNELGYEVLPHPPYSPDLLPTNYHVFKHLNNFLQGKRFHNQQ
DAENAFQEFVESQSTDFYATGINQLISRWQKCVDCNGSYFD ABD15132.1 Nb.BsrDI 36
>gi|86757493|gb|ABD15132.1| Nb.BSrDI [Geobacillus
stearothermophilus]
MTEYDLHLYADSFHEGHWCCENLAKIAQSDGGKHQIDYLQGFIPRHSLIFSDLIINITVFGSYKSWKHLP
KQIKDLLFWGKPDFIAYDPKNDKILFAVEETGAVPTGNQALQRCERIYGSARKQIPFWYLLSEFGQHKDG
GTRRDSIWPTIMGLKLTQLVKTPSIILHYSDINNPEDYNSGNGLKFLFKSLLQIIINYCTLKNPLKGMLE
LLSIQYENMLEFIKSQWKEQIDFLPGEEILNTKTKELARMYASLAIGQTVKIPEELFNWPRTDKVNFKSP
QGLIKYDELCYQLEKAVGSKKAYCLSNNAGAKPQKLESLKEWINSQKKLFDKAPKLTPPAEFNMKLDAFP
VTSNNNYYVTTSKNILYLFDYWKDLRIAIETAFPRLKGKLPTDIDEKPALIYICNSVKPGRLFGDPFTGQ
LSAFSTIFGKKNIDMPRIVVAYYPHQIYSQALPKNNKSNKGITLKKELTDFLIFHGGVVVKLNEGKAY
ABD15133.1 BsrDI A 37 gi|86757494|gb|ABD15133.1| BsrDI A
[Geobacillus stearothermophilus]
MTDYRYSFELSEEIARWAFEIKTKNTDWFVAFSNPTAGPWKRVMAIDKASNREGEVHRFGREDERPDIIL
VNDNISLILILEAKEKLNQLISKSQVDKSVDVFLTLSSILKEKSDNNYWGDRTKYINVLGILWGSEQETS
QKDIDNAFRVYRDSLVKNLKEINPTPTNICTDILVGVESIKNKKEEISIKIHVSNIYAEIYPKFTGKHLL
EKLAVLN ABN42182.1 Nt.BspD6I 38 gi|125396996|gb|ABN42182.1|
heterodimeric restriction endonuclease (R.BspD6I R.BspD6I large
subunit [Bacillus sp. D6] large
MAKKVNWYVSCSPRSPEKIQPELKVLANFEGSYWKGVKGYKAQEAFAKELAALPQFLGTTYKKEAA-
FSTR subunit)
DRVAPMKTYGFVFVDEEGYLRITEAGKMLANNRRPKDVFLKQLVKWQYPSFQHKGKEYPEEEW-
SINPLVF
VLSLLKKVGGLSKLDIAMFCLTATNNNQVDEIAEEIMQFRNEREKIKGQNKKLEFTENYFFKRFEKIYGN
VGKIREGKSDSSHKSKIETKMANARDVADATTRYFRYTGLEVARGNQLVLNPEKSDLIDEIISSSKVVKN
YTRVEEFHEYYGNPSLPQFSFETKEQLLDLAHRIRDENTRLAEQLVEHFPNVRVEIQVLEDIYNSLNKKV
DVETLKDVIYHAKELQLELKKKKLQADFNDPRQLEEVIDLLEVYHEKKNVIEEKIKARFIANKNTVFEWL
TWNGFIILGNALEYKNNFVIDEELQPVTHAAGNQPDMEIIYEDFIVLGEVTTSKGATQFKMESEPVTRHY
LNKKKELEKQGVEKELYCLFIAPEINKNTFEEFMKYNIVQNTRIIPLSLKQFNMLLMVQKKLIEKGRRLS
SYDIKNLMVSLYRTTIECERKYTQIKAGLEETLNNWVVDKEVRF ABN42183.1 ss.BspD6I
39 >gi|125396997|gbABN42183.1| heterodimeric restriction
endonuclease (R.BspD6I R.BspD6I small subunit [Bacillus sp. D6]
small
MQDILDFYEEVEKTINPPNYFEWNTYRVFKKLGSYKNLVPNFKLDDSGHPIGNAIPGVEDILVEYE-
HFSI subunit)
LIECSLTIGEKQLDYEGDSVVRHLQEYKKKGIEAYTLFLGKSIDLSFARHIGFNKESEPVIPL-
TVDQFKK LVTQLKGDGEHFNPNKLKEILIKLLRSDLGYDQAEEWLTFIEYNLK AAK27215.1
R.PleI 40 >gi|13448813|gb|AAK27215.1|AF355461_2 restriction
endonuclease R.PleI [Paucimonas lemoignei]
MAKPIDSKVLFITTSPRTPEKMVPEIELLDKNFNGDVWNKDTQTAFMKILKEESFFDGEGKNDPAFSARD
RINRAPKSLGFVILTPKLSLTDAGVELIKAKRKDDIFLRQMLKFQLPSPYHKLSDKAALFYVKPYLEIFR
LVRHFGSLTFDELMIFGLQIIDFRIFNQIVDKIEDFRVGKIENKGRYKTYKKERFEEELGKIYKDELFGL
TEASAKTLITKKGNNMRDYADACVRYLRATGMVNVSYQGKSLSIVQEKKEEVDFFLKNTEREPCFINDEA
SYVSYLGNPNYPKLFVDDVDRIKKKLRFDFKKTNKVNALTLPELKEELENEILSRKENILKSQISDIKNF
KLYEDIQEVFEKIENDRTLSDAPLMLEWNTWRAMTMLDGGEIKANLKFDDFGSPMSTAIGNMPDIVCEYD
DEQLSVEVTMASGQKQYEMEGEPVSRHLGKLKKSSEKPVYCLFIAPKINPSSVANFFMSHKVDIEYYGGK
SLIIPLELSVFRKMIEDTFKASYIPKSDNVHKLFKNFASIADEAGNEKVWYEGVKRTAMNWLSLS
AAK39546.1 MlyI 41 >gi|13786046|gb|AAK39546.1|AF355462_2 MlyIR
[Micrococcus lylae]
MASLSKTKHLFGFTSPRTIEKIIPELDILSQQFSGKVWGENQINFFDAIFNSDFYEGTTYPQDPALAARD
RITRAPKALGFIQLKPVIQLTKAGNQLVNQKRLPELFTKQLLKFQLPSPYHTQSPTVNFNVRPYLELLRL
INELGSISKTEIALFFLQLVNYNKFDEIKNKILKFRETRKNNRSVSWKTYVSQEFEKQISIIFADEVTAK
NFRTRESSDESFKKFVKTKEGNMKDYADAFFRYIRGTQLVTIDKNLHLKISSLKQDSVDFLLKNTDRNAL
NLSLMEYENYLFDPDQLIVLEDNSGLINSKIKQLDDSINVESLKIDDAKDLLNDLEIQRKAKTIEDTVNH
LKLRSDIEDILDVFAKIKKADVPDVPLFLEWNIWRAFAALNHTQAIEGNFIVDLDGMPLNTAPGKKPDIE
INYGSFSCIVEVPMSSGETQFNMEGSSVPRHYGDLVRKVDHDAYCIFIAPKVAPGTKAHFFNLNRLSTKH
YGGKTKIIPMSLDDFICFLQVGITHNFQDINKLKNWLDNLINFNLESEDEEIWFEEIISKISTWAI
YP_ AlwI 42 >gi|319768594|ref|YP_004134094.1| restriction
endonuclease, type II, 004134094.1 AlwI [Geobacillus sp. Y412MC52]
MNKKNTRKVWFITRPERDPRFHQEALLALQKATDDFRLKWAGNREVEKRYEEELANMGIKRNNVSHDGSG
GRTWMAMLKTFSYCYVDDDGYIRLTKVGEKLIQGEKVYENTRKQVLTLQYPNAYFLEPGFRPKFDEGFRI
RPVLFLIKLANDERLDFYVTKEEITYFAMTAQKDSQLDEIVHKILAFRKAGPREREEMKQDIAAKFDHRE
RSDKGARDFYEAHSDVAHTFMLISDYTGLVEYIRGKALKGDSSKINEIKQEIAEIEKRYPFNTRYMISLE
RMAENSGLDVDSYKASRYGNIKPAANSSKLRAKAERILAQFPSIESMSKEEIAGALQKYLSPRDIEKVIH
EIVENKDDFEGINSDFVETYLNEKDNLAFEDKTGQIFSALGFDVAMRPKAKNGERTEIEIIARYGGSKFG
IIDAKNYAGKFPLSSSLVSHMASEYIPNYTGYEGKELTFFGYVTANDFSGERNLEKISDKAKRITGNPIS
GFLVTARTLLGFLDYCIENDVPLEDRAELFVKAVKNKGYKSLEALLRELKETI AAY97906.1
Mva1269I 43 >gi|68480350|gb|AAY97906.1| Mva1269I restriction
endonuclease [Kocuria varians]
MYLNTAVFNIYGDNIVECSRAFHYILEGFKLANISITQEYDLQNITTPKFCIYTDKFRYIFIFIPGTSAS
RWNKDIYKELVLNNGGPLKEGADAIITRIFSEDSELVLASMEFSAALPAGNNTWQRSGRAYSLTAANIPY
FYIVQLGGKEIKKGKDGKSDKFATRLPNPALSLSFTLNTIKKPAPSLIVYDQAPEADSAISDLYSNCYGI
DDFSLYLFKLITEENNLHELKNIYNKNVEFLQLRSVDEKGKNFSGKDYKYIFEHKDPYKGLTEVVKERKI
PWKKKTATKTFENFPLRNQAPIFRLIDFLSTKSYGIVSKDSLPLTFIPSEHRVEVANYICNQLYIDKVSD
EFVKWIYKKEDLAICIINGFKPGGDDSRPDRGLPPFTKMLTNLDILTLMFGPAPPTQWDYLDSDPEKLNK
TNGLWQSIFAFSDAILVDSSTRDNNKFVYNAYLKEHWVVQREKKESNTPISYFPKSVGEHDVDTSLHILF
TYIGKHFESACNPPGGDWSGVSLLKNNIEYRWTSMYRVSQDGTKRPDHIYQLVYNSTDTLLLIESKGIKN
DLLKSKEANVGIGMINYLKNLMARDYTAVKKDGEWKNIHGQMTLDKFLTFSAVAYLFTTDFDNEYTSAAE
LLVHSNTQLAFALEIKEKNSVMHIFTANTVAYNFAEYLLETMRNSHLPLKIYKPI ADR72996.1
BsrI 44 >gi|313667100|gb|ADR72996.1| BsrI [Geobacillus
stearothermophilus]
MRNIRIYSEVKEQGIFFKEVIQSVLEKANVEVVLVNSAMLDYSDVSVISLIRNQKKFDLLVSEVRDKREI
PIVMVEFSTAVTTDDHELQRADAMFWAYKYKIPYLKISPMEKKSQTADDKFGGGRLLSVNDQIIHMYRTD
GVMYHIEWESMDNSAYVKNAELYPSCPDCAPELASLFRCLLETIEKCENIEDYYRILLDKLGKQKVAVKW
GNFREEKTLEQWKHEKFDLLERFSKSSSRMEYDKDKKELKIKVNRYGHAMDPERGILAFWKLVLGDEWKI
VAEFQLQRKTLKGRQSYQSLFDEVSQEEKLMNIASEIIKNGNVISPDKAIEIHKLATSSTMISTIDLGTP
ERKYITDDSLKGYLQHGLITNIYKNLLYYVDEIRFTDLQRKTIASLTWNKEIVNDYYKSLMDQLLDKNLR
VLPLTSIKNISEDLITWSSKEILINLGYKILAASYPEAQGDRCILVGPTGKKTERKFIDLIAISPKSKGV
ILLECKDKLSKSKDDCEKMNDLLNHNYDKVTKLINVLNINNYNYNNIIYTGVAGLIGRKNVDNLPVDFVI
KFKYDAKNLKLNWEINSDILGKHSGSFSMEDVAVVRKRS AAL86024.1 BsmI 45
>gi|19347662|gb|AAL86024.1| BsmI [Geobacillus
stearothermophilus]
MNVFRINGDNIIECERVIDLILSKINPQKVKRGFISLSCPFIEIIFKEGHDYFHWRFDMFPGFNKNTNDR
WNSNILDLLSQKGSFLYETPDVIITSLNNGKEEILMAIEFCSALQAGNQAWQRSGRAYSVGRTGYPYIYI
VDFVKYELNNSDRSRKNLRFPNPAIPYSYISHSKNTGNFIVQAYFRGEEYQPKYDKKLKFFDETIFAEDD
IADYIIAKLQHRDTSNIEQLLINKNLKMVEFLSKNTKNDNNFTYSEWESIYNGTYRITNLPSLGRFKFRK
KIAEKSLSGKVKEENNIVQRYSVGLASSDLPFGVIRKESANDFINDVCKLYNINDMKIIKELKEDADLIV
CMLKGFKPRGDDNRPDRGALPLVAMLAGENAQIFTFIYGPLIKGAINLIDQDINKLAKRNGLWKSFVSLS
DFIVLDCPIIGESYNEFRLIINKNNKESILRKTSKQQNILVDPTPNHYQENDVDTVIYSIFKYIVPNCFS
GMCNPPGGDWSGLSIIRNGHEFRWLSLPRVSENGKRPDHVIQILDLFEKPLLLSIESKEKPNDLEPKIGV
QLIKYIEYLFDFTPSVQRKIAGGNWEFGNKSLVPNDFILLSAGAFIDYDNLTENDYEKIFEVTGCDLLIA
IKNQNNPQKWVIKFKPKNTIAEKLVNYIKLNFKSNIFDTGFFHIEG ADI24225.1 Nb.BtsCI
46 >gi|297185870|gb|AD124225.1| BtsCI bottom-strand nicking
enzyme variant [synthetic construct]
MKRILYLLTEERPKINIIHQIINLEYKATLHFGAKIVPVMNEENKFTFIYHVKGIEVEGFDAVLIKIVSG
HSSFVDYLVFDSNDLKPEKNTITLFDLDQYELDLSYYFGKGWIVRIPSPSDLPKYVVEETKTDDHESRNT
NAYQRSSKFVFCELYYGKEVKKYMLYDISDGRTLSGTDTHNFGMRMLVTNNVNLVGVPNMYLPFTDIKEF
INEKNRIADNGPSHNVPIRLKLDKEKNVIYISAKLDKGNGKNKNKISNDPNIGAVAIISATLANLNWKGD
IEIINHNLLPSSISSRSNGNKLLYIMKKLGVRFNNINVNWNNIKNNINYFFYNITSEKIVSIYYHLYVED
KLSNARVIFDNHAGCGKSYFRTLNNKIIPVGKEIPLPALVIFDSDQNIVKVIAAAKAENVYNGVEQLSTF
DKFIESYINKYYPGAAVECSVITWGKSSNPYVSFYLDKDGSAVFL ADI242241 Nt.BtsCI 47
>gi|297185868|gb|ADI24224.1| BtsCI top-strand nicking enzyme
variant [synthetic construct]
MKRILYLLTEERPKINIIHQIINLEYKATLHFGAKIVPVMNEENKFTFIYHVKGIEVEGFDAVLIKIVSG
HSSFVDYLVFDSNDLKPEKNTITLFDLDQYELDLSYYFGKGWIVRIPSPSDLPKYVVFETKTDDHESRNT
NAYQRSSKFVFCELYYGKEVKKYMLYDISDGRTLSGTDTHNFGMRMLVTNNVNLVGVPNMYLPFTDIKEF
INEKNRIADNGPSHNVPIRLKLDKEKNVIYISAKLDKGNGKNKNKISNDPNIGAVAIISATLRNLNWKGD
IEIINENLLPSSISSRSNGNKLLYIMKKLGVRFNNINVNWNNIKNNINYFFYNITSEKIVSIYYHLYVED
KLSNARVIFDNHAGCGKSYFRTLNNKIIPVGKEIPLPDLVIFDSDQNIVKVIEAEKAENVYNGVEQLSTF
DKFIESYINKYYPGAAVECSVITWGKSSNPYVSFYLDKDGSAVFL
>gi|85720924|gb|ABC75874.1| R1.BtsI [Geobacillus
thermoglucosidasius]
MKITEGIVHVAMRHFLKSNGWKLIAGQYPGGSDDELTALNIVDPVVARDNSPDPRRHSLGKIVPDLIAYK
NDDLLVIEAKPKYSQDDRDKLLYLLSERKHDFYAALEKFATERNHPELLPVSKLNIIPGLAFSASENKFK
KDPGFVYIRVSGIFEAFMEGYDWG ABC758741 R1.BtsI 48
>gi|85720924|gb|ABC75874.1| R1.BtsI [Geobacillus
thermoglucosidasius]
MKITEGIVHVAMRHFLKSNGWKLIAGQYPGGSDDELTALNIVDPVVARDNSPDPRRHSLGKIVPDLIAYK
NDDLLVIEAKPKYSQDDRDKLLYLLSERKHDFYAALEKFATERNHPELLPVSKLNIIPGLAFSASENKFK
KDPGFVYIRVSGIFEAFMEGYDWG ABC75876.1 R2.BtsI 49
>gi|85720926|gb|ABC75876.1| R2.BtsI [Geobacillus
thermoglucosidasius]
MQIEQLMKSLTIYFDDIQEGLWFKNLHPLLESASLEAITGSLKRNPNLADVLKYDRPDIILTLNQTPILV
IERTIEVPSGHNVGQRYGRLAAASEAGVPLVYFGPYAARKHGGATEGPRYMNLRLFYALDVMQKVNGSAI
TTINWPVDQNFEILQDPSKDKRMKEYLEMFFDNLLKYGIAGINLAIRNSSFQAEQLAEREKFVETMITNP
EQYDVPPDSVQILNAERFFNELGISENKRIICDEVVLYQVGMTYVRSDPYTGMALLYKYLYILGSERNRC
LILKFPNITTDMWKKVAFGSRERKDVRIYRSVSDGILFADGYLSKEEL AAX146521 BbvCI 50
>gi|60202520|gb|AAX14652.1| BbvCI endonuclease subunit 1 subunit
1 [Brevibacillus brevis]
MINEDFFIYEQLSHKKNLEQKGKNAFDEETEELVRQAKSGYHAFIEGINYDEVTKLDLNSSVAALEDYIS
IAKEIEKKHKMFNWRSDYAGSIIPEFLYRIVHVATVKAGLKPIFSTRNTIIEISGAAHREGLQIRRKNED
FALGFHEVDVKIASESHRVISLAVACEVKTNIDKNKLNGLDFSAERMKRTYPGSAYFLITETLDFSPDEN
HSSGLIDEIYVLRKQVRTKNRVQKAPLCPSVFAELLEDILEISYRASNVKGHVYDRLEGGKLIRV
AAX146531 BbvCI 51 >gi|60202521|gb|AAX14653.1| BbvCI
endonuclease subunit 2 subunit 2 [Brevibacillus brevis]
MFNQFNPLVYTHGGKLERKSKKDKTASKVFEEFGVMEAYNCWKEASLCIQQRDKDSVLKLVAALNTYKDA
VEPIFDSRLNSAQEVLQPSILEEFFEYLFSRIDSIVGVNIPIRHPAKGYLSLSFNPHNIETLIQSPEYTV
RAKDHDFIIGGSAKLTIQGHGGEGETTNIVVPAVAIECKRYLERNMLDECAGTAERLKRATPYCLYFVVA
EYLKLDDGAPELTEIDEIYILRHQRNSERNKPGFKPNPIDGELIWDLYQEVMNHLGKIWWDPNSALQRGK
VFNRP CAA74998.1 Bpu10I alpha 52 >gi|2894388|emb|CAA74998.1|
Bpu10I restriction endonuclease alpha subunit subunit [Bacillus
pumilus]
MGVEQEWIKNITDMYQSPELIPSHASNLLHQLKREKRNEKLKKALEIITPNYISYISILLNNHNMTRKEI
VILVDALNEYMNTLRHPSVKSVFSHQADFYSSVLPEFFNLLFRNLIKGLNEKIKVNSQKDIIIDCIFDPY
NEGRVVFKKKRVDVAIILKNKFVFNNVEISDFAIPLVAIEIKTNLDKNMLSGIEQSVDSLKETFPLCLYY
CITELADFAIEKQNYASTRIDEVFILRKQKRGPVARGTPLEVVHADLILEVVEQVGEHLSKFKDPIKTLK
ARMTEGYLIKGKGK CAA74999.1 Bpu10I beta 53
>gi|2894389|emb|CAA74999.1| Bpu10I restriction endonuclease beta
subunit subunit [Bacillus pumilus]
MTQIDLSNTKHGSILFEKQKNVKEKYLQQAYKHYLYFRRSIDGLEITNDEAIFKLTQAANNYRDNVLYLF
ESRPNSGQEAFRYTILEEFFYHLFKDLVKKKFNQEPSSIVMGKANSYVSLSFSPESFLGLYENPIPYIHT
KDQDFVLGCAVDLKISPKNELNKENETEIVVPVIAIECKTYIERNMLDSCAATASRLKAAMPYCLYIVAS
EYMKMDQAYPELTDIDEVFILCKASVGERTALKKKGLPPRKLDENLMVELFHMVERHLNRVWWSPNEALS
RGRVIGRP ABM69266.1 BmrI 54 >gi|123187377|gb|ABM69266.1| BmrI
[Bacillus megaterium]
MNYFSLHPNVYATGRPKGLINMLESVWISNQKPGDGTMYLISGFANYNGGIRFYETFTEHINHGGKVIAI
LGGSTSQRLSSKQVVAELVSRGVDVYIINRKRLLHAKLYGSSSNSGESLVVSSGNFTGPGMSQNVEASLL
LDNNTTSSMGFSWNGMVNSMLDQKWQIHNLSNSNPTSPSWNLLYDERTTNLTLDDTQKVTLILTLGHADT
ARIQAAPKSKAGEGSQYFWLSKDSYDFFPPLTIRNKRGTKATYSCLINMNYLDIKYIDSECRVTFEAENN
FDFRLGTGKLRYTNVAASDDIAAITRVGDSDYELRIIKKGSSNYDALDSAAVNFIGNRGKRYGYIPNDEF
GRIIGAKF CAC12783.1 BfiI 55 >gi|10798463|emb|CAC12783.1|
restriction endonuclease BfiI [Bacillus firmus]
MNFFSLHPNVYATGRPKGLIGMLENVWVSNHTPGEGTLYLISGFSNYNGGVRFYETFTEHINQGGRVIAI
LGGSTSQRLSSRQVVEELLNRGVEVHIINRKRILHAKLYGTSNNLGESLVVSSGNFTGPGMSQNIEASLL
LDNNTTQSMGFSWNDMISEMLNQNWHIHNMTNATDASPGWNLLYDERTTNLTLDETERVTLIVTLGHADT
ARIQAAPGTTAGQGTQYFWLSKDSYDFFPPLTIRNRRGTKATYSSLINMNYIDINYTDTQCRVTFEAENN
FDFRLGTGKLRYTGVAKSNDIAAITRVGDSDYELRIIKQGTPEHSQLDPYAVSFIGNRGKRFGYISNEEF
GRIIGVTF P05725.1 1-CreI 1 >gi|140470|sp|P05725.1|DNE1_CHLRE
RecName: Full = DNA endonuclease I- CreI; AltName: Full = 23S rRNA
intron protein
MNTKYNKEFLLYLAGFVDGDGSIIAQIKPNQSYKFKHQLSLAFQVTQKTQRRWFLDKLVDEIGVGYVRDR
GSVSDYILSEIKPLHNFLTQLQPFLKLKQKQANLVLKIIWRLPSAKESPDKFLEVCTWVDQIAALNDSKT
RKTTSETVRAVLDSLSEKKKSSP Q9UQ84.2 ExoI 56
>gi|85700954|sp|Q9UQ84.2|EXO1_HUMAN RecName: Full = Exonuclease
1; (EXO1_HUMAN) Short = hExo1; AltName: Full = Exonuclease I; Short
= hExoI
MGIQGLLQFIKEASEPIHVRKYKGQVVAVDTYCWLHKGAIACAEKLAKGEPTDRYVGFCMKFVNMLLSHG
IKPILVFDGCTLPSKKEVERSRRERRQANLLKGKQLLREGKVSEARECFTRSINITHAMAHKVIKAARSQ
GVDCLVAPYEADAQLAYLNKAGIVQAIITEDSDLLAFGCKKVILKMDQFGNGLEIDQARLGMCRQLGDVF
TEEKFRYMCILSGCDYLSSLRGIGLAKACKVLRLANNPDIVKVIKKIGHYLKMNITVPEDYINGFIRANN
TFLYQLVFDPIKRKLIPLNAYEDDVDPETLSYAGQYVDDSIALQIALGEKDINTFEQIDDYNPDTAMPAH
SRSHSWDDKTCQKSANVSSIWHRNYSPRPESGTVSDAPQLKENPSTVGVERVISTKGLNLPRKSSIVKRP
RSAELSEDDLLSQYSLSETKKTKKNSSEGNKSLSFSEVFVPDLVNGPTNKKSVSTPPRTRNKFATFLQRK
NEESGAVVVPGTRSRFFCSSDSTDCVSNKVSIQPLDETAVTDKENNLHESEYGDQEGKRLVDTDVARNSS
DDIPNNHIPGDHIPDKATVFTDEESYSFESSKFTRTISPPTLGTLRSCFSWSGGLGDFSRTPSPSPSTAL
QQFRRKSDSPTSLPENNMSDVSQLKSEESSDDESHPLREEACSSQSQESGEFSLQSSNASKLSQCSSKDS
DSEESDCNIKLLDSQSDQTSKLRLSHFSKKDTPLRNKVPGLYKSSSADSLSTTKIKPLGPARASGLSKKP
ASIQKRKHHNAENKPGLQIKLNELWKNFGFKKDSEKLPPCKKPLSPVRDNIQLTPEAEEDIFNKPECGRV
QRAIFQ P39875.2 Yeast ExoI 57 >gi|1706421|sp|P39875.2|EXO1_YEAST
RecName: Full = Exodeoxyribo- (EXO1_YEAST) nuclease 1; AltName:
Full = Exodeoxyribonuclease I; Short = EXO I; Short = Exonuclease
I; AltName: Full = Protein DHS1
MGIQGLLPQLKPIQNPVSLRRYEGEVLAIDGYAWLHRAACSCAYELAMGKPTDKYLQFFIKRFSLLKTFK
VEPYLVEDGDAIPVKKSTESKARDKRKENKAIAERLWACGEKKNAMDYFQKCVDITPEMAKCIICYCKLN
GIRYIVAPFEADSQMVYLEQKNIVQGIISEDSDLLVFGCRRLITKLNDYGECLEICRDNFIKLPKKFPLG
SLTNEEIITMVCLSGCDYTNGIPKVGLITAMKLVRRFNTIERIILSIQREGKLMIPDTYINEYEAAVLAF
QFQRVFCPIRKKIVSLNEIPLYLKDTESKRKRLYACIGFVIHRETQKKQIVHFDDDIDHHLHLKIAQGDL
NPYDFHQPLANREHKLQLASKSNIEFGKTNTTNSEAKVKPIESFFQKMTKLDHNPKVANNIHSLRQAEDK
LTMAIKRRKLSNANVVQETLKDTRSKFFNKPSMTVVENFKEKGDSIQDFKEDTNSQSLEEPVSESQLSTQ
IPSSFITTNLEDDDELSEEVSEVVSDIEEDRKNSEGKTIGNEIYNTDDDGDGDTSEDYSETAESRVPTSS
TTSFPGSSQRSISGCTKVLQKFRYSSSFSGVNANRQPLFPRHVNQKSRGMVYVNQNRDDDCDDNDGKNQI
TQRPSLRKSLIGARSQRIVIDMKSVDERKSFESSPILHEESKKRDIETTKSSQARPAVRSISLLSQFVYK
GK BAJ43803.1 E.coli ExoI 58 >gi|315136644|dbj|BAJ43803.1|
exonuclease I [Escherichia coli DH1]
MMNDGKQQSTFLFHDYETFGTHPALDRPAQFAAIRTDSEFNVIGEPEVFYCKPADDYLPQPGAVLITGIT
PQEARAKGENEAAFAARIHSLFTVPKTCILGYNNVRFDDEVTRNIFYRNFYDPYAWSWQHDNSRWDLLDV
MRACYALRPEGINWPENDDGLPSFRLEHLTKANGIEHSNAHDAMADVYATIAMAKLVKTRQPRLFDYLFT
HRNKHKLMALIDVPQMKPLVHVSGMFGAWRGNTSWVAPLAWHPENRNAVIMVDLAGDISPLLELDSDTLR
ERLYTAKTDLGDNAAVPVKLVHINKCPVLAQANTLRPEDADRLGINRQHCLDNLKILRENPQVREKVVAI
FAEAEPFTPSDNVDAQLYNGFFSDADRAAMKIVLETEPRNLPALDITFVDKRIEKLLFNYRARNFPGTLD
YAEQQRWLEHRRQVFTPEFLQGYADELQMLVQQYADDKEKVALLKALWQYAEEIV
Q913Q50.1 Human TREX2 59 >gi|47606206|sp|Q913Q50.1|TREX2_HUMAN
RecName: Full = Three prime repair exonuclease 2; AltName: Full =
3'-5' exonuclease TREX2
MGRAGSPLPRSSWPRMDDCGSRSRCSPTLCSSLRTCYPRGNITMSEAPRAETFVFLDLEATGLPSVEPEI
AELSLFAVHRSSLENPEHDESGALVLPRVLDKLTLCMCPERPFTAKASEITGLSSEGLARCRKAGFDGAV
VRTLQAFLSRQAGPICLVAHNGFDYDFPLLCAELRRLGARLPRDTVCLDTLPALRGLDRAHSHGTRARGR
QGYSLGSLFHRYFRAEPSAAHSAEGDVHTLLLIFLHRAAELLAWADEQARGWAHIEPMYLPPDDPSLEA
Q91XB0.2 Mouse TREX1 60 >gi|47606196|sp|Q91XB0.2|TREX1_MOUSE
RecName: Full = Three prime repair exonuclease 1; AltName: Full =
3'-5' exonuclease TREX1
MGSQTLPHGHMQTLIFLDLEATGLPSSRPEVTELCLLAVHRRALENTSISQGHPPPVPRPPRVVDKLSLC
IAPGKACSPGASEITGLSKAELEVQGRQRFDDNLAILLRAFLQRQPQPCCLVAHNGDRYDFPLLQTELAR
LSTPSPLDGTFCVDSIAALKALEQASSPSGNGSRKSYSLGSIYTRLYWQAPTDSHTAEGDVLTLLSICQW
KPQALLQWVDEHARPFSTVKPMYGTPATTGTTNLRPHAATATTPLATANGSPSNGRSRRPKSPPPEKVPE
APSQEGLLAPLSLLTLLTLAIATLYGLFLASPGQ Q9NSU2.1 Human TREX1 61
>gi|47606216|sp|Q9NSU2.1|TREX1_HUMAN RecName: Full = Three prime
repair exonuclease 1; AltName: Full = 3'-5' exonuclease TREX1;
AltName: Full = DNase III
MGPGARRQGRIVQGRPEMCFCPPPTPLPPLRILTLGTHTPTPCSSPGSAAGTYPTMGSQALPPGPMQTLI
FEDMEATGLPFSQPKVTELCLLAVHRCALESPPTSQGPPPTVPPPPRVVDKLSLCVAPGKACSPAASEIT
GLSTAVLAAHGRQCFDDNLANLLLAFLRRQPQPWCLVAHNGDRYDFPLLQAELAMLGLTSALDGAFCVDS
ITALKALERASSPSEHGPRKSYSLGSIYTRLYGQSPPDSHTAEGDVLALLSICQWRPQALLRWVDAHARP
FGTIRPMYGVTASARTKPRPSAVTTTAHLATTRNTSPSLGESRGTKDLPPVKDPGALSREGLLAPLGLLA
ILTLAVATLYGLSLATPGE Q9BG99.1 Bovine TREX1 62
>gi|47606205|sp|Q9BG99.1|TREX1_BOVIN RecName: Full = Three prime
repair exonuclease 1; AltName: Full = 3'-5' exonuclease TREX1
MGSRALPPGPVQTLIFLDLEATGLPFSQPKITELCLLAVHRYALEGLSAPQGPSPTAPVPPRVLDKLSLC
VAPGKVCSPAASEITGLSTAVLAAHGRRAFDADLVNLIRTFLQRQPQPWCLVAHNGDRYDFPLLRAELAL
LGLASALDDAFCVDSIAALKALEPTGSSSEHGPRKSYSLGSVYTRLYGQAPPDSHTAEGDVLALLSVCQW
RPRALLRWVDAHAKPFSTVKPMYVITTSTGTNPRPSAVTATVPLARASDTGPNLRGDRSPKPAPSPKMCP
GAPPGEGLLAPLGLLAFLTLAVAMLYGLSLAMPGQ AAH91242.1 Rat TREX1 63
>gi|60688197|gb|AAH91242.1| Trex1 protein [Rattus norvegicus]
MGSQALPHGHMQTLIFLDLEATGLPYSQPKITELCLLAVHRHALENSSMSEGQPPPVPKPPRVVDKLSLC
IAPGKPCSSGASEITGLTTAGLEAHGRQRFNDNLATLLQVFLQRQPQPCCLVAHNGDRYDEPLLQAELAS
LSVISPLDGTFCVDSIAALKTLEQASSPSEHGPRKSYSLGSIYTRLYGQAPTDSHTAEGDVLALLSICQW
KPQALLQWVDKHARPFSTIKPMYGMAATTGTASPRLCAATTSSPLATANLSPSNGRSRGKRPTSPPPENV
PEAPSREGLLAPLGLLTFLTLAIAVLYGIFLASPGQ AAH63664.1 Human DNA2 64
>gi|39793966|gb|AAH63664.1| DNA2 protein [Homo sapiens]
FAIPASRMEQLNELELLMEKSFWEEAELPAELFQKKVVASFPRTVLSTGMDNRYLVLAVNTVQNKEGNCE
KRLVITASQSLENKELCILRNDWCSVPVEPGDIIHLEGDCTSDTWIIDKDFGYLILYPDMLISGTSIASS
IRCMRRAVLSETFRSSDPATRQMLIGTVLHEVFQKAINNSFAPEKLQELAFQTIQEIRHLKEMYRLNLSQ
DEIKQEVEDYLPSFCKWAGDFMHKNTSTDFPQMQLSLPSDNSKDNSTCNIEVVKPMDIEESIWSPRFGLK
GKIDVTVGVKIHRGYKTKYKIMPLELKTGKESNSIEHRSQVVLYTLLSQERRADPEAGLLLYLKTGQMYP
VPANHLDKRELLKLRNQMAFSLFHRISKSATRQKTQLASLPQIIEEEKTCKYCSQIGNCALYSRAVEQQM
DCSSVPIVMLPKIEEETQHLKQTHLEYFSLWCLMLTLESQSKDNKKNHQNIWLMPASEMEKSGSCIGNLI
RMEHVKIVCDGQYLHNFQCKHGAIPVTNLMAGDRVIVSGEERSLFALSRGYVKEINMTTVTCLLDRNLSV
LPESTLFRLDQEEKNCDIDTPLGNLSKLMENTFVSKKLRDLIIDFREPQFISYLSSVLPHDAKDTVACIL
KGLNKPQRQAMKKVLLSKDYTLIVGMPGTGKTTTICTLVPAPEQVEKGGVSNVTEAKLIVELTSIFVKAG
CSPSDIGIIAPYRQQLKIINDLLARSIGMVEVNTVDKYQGRDKSIVLVSFVRSNKDGTVGELLKDWRRLN
VAITRAKHKLILLGCVPSLNCYPPLEKLLNHLNSEKLIIDLPSREHESLCHILGDFQRE
P38859.1 Yeast DNA2 65 >gi|731738|sp|P38859.1|DNA2_YEAST
RecName: Full = DNA replication ATP- (DNA2_YEAST) dependent
helicase DNA2
MPGTPQKNKRSASISVSPAKKTEEKEIIQNDSKAILSKQTKRKKKYAFAPINNLNGKNTKVSNASVLKSI
AVSQVRNTSRTKDINKAVSKSVKQLPNSQVKPKREMSNLSRHHDFTQDEDGPMEEVIWKYSPLQRDMSDK
TTSAAEYSDDYEDVQNPSSTPIVPNRLKTVLSFTNIQVPNADVNQLIQENGNEQVRPKPAEISTRESLRN
IDDILDDIEGDLTIKPTITKFSDLPSSPIKAPNVEKKAEVNAEEVDKMDSTGDSNDGDDSLIDILTQKYV
EKRKSESQITIQGNTNQKSGAQESCGKNDNTKSRGEIEDHENVDNQAKTGNAFYENEEDSNCQRIKKNEK
IEYNSSDEFSDDSLIELLNETQTQVEPNTIEQDLDKVEKMVSDDLRIATDSTLSAYALRAKSGAPRDGVV
RLVIVSLRSVELPKIGTQKILECIDGKGEQSSVVVREPWVYLEFEVGDVIHIIEGKNIENKRLLSDDKNP
KTQLANDNLLVLNPDVLFSATSVGSSVGCLRRSILQMQFQDPRGEPSLVMTLGNIVHELLQDSIKYKLSH
NKISMEIIIQKLDSLLETYSFSIIICNEEIQYVKELVMKEHAENILYFVNKFVSKSNYGCYTSISGTRRT
QPISISNVIDIEENIWSPIYGLKGFLDATVEANVENNKKHIVPLEVKTGKSRSVSYEVQGLIYTLLLNDR
YEIPIEFFLLYFTRDKNMTKFPSVLHSIKHILMSRNRMSMNFKHQLQEVFGQAQSRFELPPLLRDSSCDS
CFIKESCMVLNKLLEDGTPEESGLVEGEFEILTNHLSQNLANYKEFFTKYNDLITKEESSITCVNKELFL
LDGSTRESRSGRCLSGLVVSEVVEHEKTEGAYIYCFSRRANDNNSQSMLSSQIAANDEVIISDEEGHFCL
CQGRVQFINPAKIGISVKRKLLNNRLLDKEKGVTTIQSVVESELEQSSLIATQNLVTYRIDKNDIQQSLS
LARFNLLSLFLPAVSPGVDIVDERSKLCRKTKRSDGGNEILRSLLVDNRAPKFRDANDDPVIPYKLSKDT
TLNLNQKEAIDKVMRAEDYALILGMPGTGKTTVIAEIIKILVSEGKRVLLTSYTHSAVDNILIKLRNTNI
SIMRLGMKHKVHPDTQKYVPNYASVKSYNDYLSKINSTSVVATTCLGINDILFTLNEKDFDYVILDEASQ
ISMPVALGPLRYGNRFIMVGDHYQLPPLVKNDAARLGGLEESLFKTFCEKHPESVAELTLQYRMCGDIVT
LSNFLIYDNKLKCGNNEVFAQSLELPMPEALSRYRNESANSKQWLEDILEPTRKVVFLNYDNCPDIIEQS
EKDNITNHGEAELTLQCVEGMLLSGVPCEDIGVMTLYRAQLRLLKKIFNKNVYDGLEILTADQFQGRDKK
CIIISMVRRNSQLNGGALLKELRRVNVAMTRAKSKLIIIGSKSTIGSVPEIKSFVNLLEERNWVYTMCKD
ALYKYKFPDRSNAIDEARKGCGKRTGAKPITSKSKFVSDKPIIKEILQEYES AAA45863.1
VP16 66 >gi|330318|gb|AAA45863.1| VP16 [Human herpesvirus 2]
MDLLVDDLFADRDGVSPPPPRPAGGPKNTPAAPPLYATGRLSQAQLMPSPPMPVPPAALFNRLLDDLGFS
AGPALCTMLDTWNEDLFSGFPTNADMYRECKFLSTLPSDVIDWGDAHVPERSPIDIRAHGDVAFPTLPAT
RDELPSYYEAMAQFFRGELRAREESYRTVLANFCSALYRYLRASVRQLHRQAHMRGRNRDLREMLRTTIA
DRYYRETARLARVLFLHLYLFLSREILWAAYAEQMMRPDLFDGLCCDLESWRQLACLFQPLMFINGSLTV
RGVPVEARRLRELNHIREHLNLPLVRSAAAEEPGAPLTTPPVLQGNQARSSGYFMLLIRAKLDSYSSVAT
SEGESVMREHAYSRGRTRNNYGSTIEGLLDLPDDDDAPAEAGLVAPRMSFLSAGQRPRRLSTTAPITDVS
LGDELRLDGEEVDMTPADALDDFDLEMLGDVESPSPGMTHDPVSYGALDVDDFEFEQMFTDAMGIDDFGG
ACM07430.1 Colicin E9 366 >gi|221185856|gb|ACM07430.1| colicin
E9 [Escherichia coli]
MSGGDGRGHNTGAHSTSGNINGGPTGIGVSGGASDGSGWSSENNPWGGGSGSGIHWGGGSGRGNGGGNGN
SGGGSGTGGNLSAVAAPVAFGFPALSTPGAGGLAVSISASELSAAIAGIIAKLKKVNLKFTPFGVVLSSL
IPSEIAKDDPNMMSKIVTSLPADDITESPVSSLPLDKATVNVNVRVVDDVKDERQNISVVSGVPMSVPVV
DAKPTERPGVFTASIPGAPVLNISVNDSTPAVQTLSPGVTNNTDKDVRPAGFTQGGNTRDAVIRFPKDSG
HNAVYVSVSDVLSPDQVKQRQDEENRRQQEWDATHPVEAAERNYERARAELNQANEDVARNQERQAKAVQ
VYNSRKSELDAANKTLADAIAEIKQFNRFAHDPMAGGHRMWQMAGLKAQRAQTDVNNKQAAFDAAAKEKS
DADAALSAAQERRKQKENKEKDAKDKLDKESKRNKPGKATGKGKPVGDKWLDDAGKDSGAPIPDRIADKL
RDKEEKSFDDFRKAVWEEVSKDPELSKNLNPSNKSSVSKGYSPFTPKNQQVGGRKVYELHHDKPISQGGE
VYDMDNIRVTTPKRHIDIHRGK NP_775816.1 APFL 367
>gi|135233|sp|P14870.1|T2F1_PLAOK RecName: Full = Type-2
restriction enzyme FokI; Short = R.FokI; AltName: Full =
Endonuclease FokI; AltName: Full = Type II restriction enzyme FokI;
AltName: Full = Type IIS restriction enzyme FokI
MFLSMVSKIRTFGWVQNPGKFENLKRVVQVFDRNSKVHNEVKNIKIPTLVKESKIQKELVAIMNQHDLIY
TYKELVGTGTSIRSEAPCDAIIQATIADQGNKKGYIDNWSSDGFLRWAHALGFIEYINKSDSFVITDVGL
AYSKSADGSAIEKEILIEAISSYPPAIRILTLLEDGQHLTKFDLGKNLGFSGESGFTSLPEGILLDTLAN
AMPKDKGEIRNNWEGSSDKYARMIGGWLDKLGLVKQGKKEFIIPTLGKPDNKEFISHAFKITGEGLKVLR
RAKGSTKFTRVPKRVYWEMLATNLTDKEYVRTRRALILEILIKAGSLKIEQIQDNLKKLGFDEVIETIEN
DIKGLINTGIFIEIKGRFYQLKDHILQFVIPNRGVTKQLVKSELEEKKSELRHKLKYVPHEYIELIEIAR
NSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQ
RYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGE
MIKAGTLTLEEVRRKFNNGEINF P14870.1 Fold 368
>gi|221185857|gb|ACM07431.1| colicin E9 immunity protein
[Escherichia coli]
MELKHSISDYTEAEFLQLVTTICNADISSEEELVKLVTHFEEMTEHPSGSDLIYYPKEGDDDSPSGIVNT
VKQWRAANGKSGFKQG
[0084] In another preferred embodiment according to the method of
the present invention, the peptidic linker that can link said
catalytic domain to the core TALE scaffold according to the method
of the present invention can be selected from the group consisting
of NFS1, NFS2, CFS1, RM2, BQY, QGPSG, LGPDGRKA, 1a8h.sub.--1,
1dnpA.sub.--1, 1d8cA.sub.--2, 1ckqA.sub.--3, 1sbp.sub.--1,
1ev7A.sub.--1, 1alo.sub.--3, 1amf.sub.--1, 1adjA.sub.--3,
1fcdC.sub.--1, 1a13.sub.--2, 1g3p.sub.--1, 1acc.sub.--3,
1ahjB.sub.--1, 1acc.sub.--1, 1af7.sub.--1, 1heiA.sub.--1,
1bia.sub.--2, 1igtB.sub.--1, 1nfkA.sub.--1, 1au7A.sub.--1, 1
bpoB.sub.--1, 1b0pA.sub.--2, 1c05A.sub.--2, 1gcb.sub.--1,
1bt3A.sub.--1, 1b3o8.sub.--2, 16vpA.sub.--6, 1dhx.sub.--1,
1b8aA.sub.--1 and 1qu6A.sub.--1, as listed in Table 3 (SEQ ID NO:
67 to SEQ ID NO: 104 and SEQ ID NO: 372 to SEQ ID NO: 415). In a
more preferred embodiment, the peptidic linker that can link said
catalytic domain to the core TALE scaffold according to the method
of the present invention can be selected from the group consisting
of NFS1 (SEQ ID NO: 98), NFS2 (SEQ ID NO: 99) and CFS1 (SEQ ID NO:
100). In the scope of the present invention is also encompassed the
case where a peptidic linker is not needed to fuse a catalytical
domain to the TALE scaffold in order to obtain a cTALEN according
to the present invention.
TABLE-US-00003 TABLE 3 List of peptidic linkers that can be used in
compact TALENs or enhanced compact TALENs. Amino SEQ ID Name (PDB)
Acids Size Length Sequence NO 1a8h_1 285-287 3 6,636 NVG 67 1dnpA_1
130-133 4 7,422 DSVI 68 1d8cA_2 260-263 4 8,782 IVEA 69 1ckqA_3
169-172 4 9,91 LEGS 70 1sbp_1 93-96 4 10,718 YTST 71 1ev7A_1
169-173 5 11,461 LQENL 72 1alo_3 360-364 5 12,051 VGRQP 73 1amf_1
81-85 5 13,501 LGNSL 74 1adjA_3 323-328 6 14,835 LPEEKG 75 1fcdC_1
76-81 6 14,887 QTYQPA 76 1al3_2 265-270 6 15,485 FSHSTT 77 1g3p_1
99-105 7 17,903 GYTYINP 78 1acc_3 216-222 7 19,729 LTKYKSS 79
1ahjB_1 106-113 8 17,435 SRPSESEG 80 1acc_1 154-161 8 18,776
PELKQKSS 81 1af7_1 89-96 8 22,502 LTTNLTAF 82 1heiA_1 322-330 9
13,534 TATPPGSVT 83 1bia_2 268-276 9 16,089 LDNFINRPV 84 1igtB_1
111-119 9 19,737 VSSAKTTAP 85 1nfkA_1 239-248 10 13,228 DSKAPNASNL
86 1au7A_1 103-112 10 20,486 KRRTTISIAA 87 1bpoB_1 138-148 11
21,645 PVKMFDRHSSL 88 1b0pA_2 625-635 11 26,462 APAETKAEPMT 89
1c05A_2 135-148 14 23,819 YTRLPERSELPAEI 90 1gcb_1 57-70 14 27,39
VSTDSTPVTNQKSS 91 1bt3A_1 38-51 14 28,818 YKLPAVTTMKVRPA 92 1b3oB_2
222-236 15 20,054 IARTDLKKNRDYPLA 93 16vpA_6 312-332 21 23,713
TEEPGAPLTTPPTLHGNQARA 94 1dhx_1 81-101 21 42,703
ARFTLAVGDNRVLDMASTYFD 95 1b8aA_1 95-120 26 31,305
IVVLNRAETPLPLDPTGKVKAELDTR 96 1qu6A_1 79-106 28 51,301
ILNKEKKAVSPLLLTTTNSSEGLSMGNY 97 NFS1 -- 20 -- GSDITKSKISEKMKGQGPSG
98 NFS2 -- 23 -- GSDITKSKISEKMKGLGPDGRKA 99 CFS1 -- 10 --
SLTKSKISGS 100 RM2 -- 32 -- AAGGSALTAGALSLTAGALSLTAGALSGGGGS 101
BQY -- 25 -- AAGASSVSASGHIAPLSLPSSPPSVGS 102 QGPSG -- 5 -- QGPSG
103 LGPDGRKA -- 8 -- LGPDGRKA 104 TAL1 -- 15 -- SGGSGSNVGSGSGSG 372
TAL2 -- 20 -- SGGSGSLTTNLTAFSGSGSG 373 TAL3 -- 22 --
SGGSGSKRRTTISIAASGSGSG 374 TAL4 -- 17 -- SGGSGSVGRQPSGSGSG 375 TAL5
-- 26 -- SGGSGSYTRLPERSELPAEISGSGSG 376 TAL6 -- 38 --
SGGSGSIVVLNRAETPLPLDPTGKVKAELDTRSGSGSG 377 TAL7 -- 21 --
SGGSGSTATPPGSVTSGSGSG 378 TAL8 -- 21 -- SGGSGSLDNFINRPVSGSGSG 379
TAL9 -- 21 -- SGGSGSVSSAKTTAPSGSGSG 380 TAL10 -- 22 --
SGGSGSDSKAPNASNLSGSGSG 381 TAL11 -- 23 -- SGGSGSPVKMFDRHSSLSGSGSG
382 TAL12 -- 23 -- SGGSGSAPAETKAEPMTSGSGSG 383 TAL13 -- 26 --
SGGSGSVSTDSTPVTNQKSSSGSGSG 384 TAL14 -- 16 -- SGGSGSDSVISGSGSG 385
TAL15 -- 33 -- SGGSGSARFTLAVGDNRVLDMASTYFDSGSGSG 386 TAL16 -- 17 --
SGGSGSLQENLSGSGSG 387 TAL17 -- 19 -- SGGSGSGYTYINPSGSGSG 388 TAL18
-- 26 -- SGGSGSYKLPAVITMKVRPASGSGSG 389 TAL19 -- 16 --
SGGSGSLEGSSGSGSG 390 TAL20 -- 16 -- SGGSGSIVEASGSGSG 391 TAL21 --
18 -- SGGSGSQTYQPASGSGSG 392 TAL22 -- 27 --
SGGSGSIARTDLKKNRDYPLASGSGSG 393 TAL23 -- 18 -- SGGSGSLPEEKGSGSGSG
394 TAL24 -- 16 -- SGGSGSYTSTSGSGSG 395 TAL25 -- 20 --
SGGSGSSRPSESEGSGSGSG 396 TAL26 -- 17 -- SGGSGSLGNSLSGSGSG 397 TAL27
-- 19 -- SGGSGSLTKYKSSSGSGSG 398 TAL28 -- 33 --
SGGSGSTEEPGAPLTTPPTLHGNQARASGSGSG 399 TAL29 -- 18 --
SGGSGSFSHSTTSGSGSG 400 TAL30 -- 20 -- SGGSGSPELKQKSSSGSGSG 401
TAL31 -- 40 -- SGGSGSILNKEKKAVSPLIITTINSSEGLSMGNYSGSGSG 402 TAL32
-- 31 -- ELAEFHARYADLLLRDLRERPVSLVRGPDSG 403 TAL33 -- 31 --
ELAEFHARPDPLLLRDLRERPVSLVRGLGSG 404 TAL34 -- 26 --
ELAEFHARYADLLLRDLRERSGSGSG 405 TAL35 -- 31 --
DIFDYYAGVAEVMLGHIAGRPATRKRWPNSG 406 TAL36 -- 31 --
DIFDYYAGPDPVMLGHIAGRPATRKRWLGSG 407 TAL37 -- 26 --
DIFDYYAGVAEVMLGHIAGRSGSGSG 408 Linker A 37
SIVAQLSRPDPALVSFQKLKLACLGGRPALDAVKKGL 409 Linker B 37
SIVAQLSRPDPAAVSAQKAKAACLGGRPALDAVKKGL 410 Linker C 37
SIVAQLSRPDPAVVTFHKLKLACLGGRPALDAVKKGL 411 Linker D 44
SIVAQLSRPDPAQSLAQELSLNESQIKIACLGGRPALDAVKKGL 412 Linker E 40
SIVAQLSRPDPALQLPPLERLTLDACLGGRPALDAVKKGL 413 Linker F 38
SIVAQLSRPDPAIHKKFSSIQMACLGGRPALDAVKKGL 414 Linker G 40
SIVAQLSRPDPAAAAATNDHAVAAACLGGRPALDAVKKGL 415
[0085] Depending from its structural composition [type of core TALE
scaffold, type of catalytic domain(s) with associated enzymatic
activities and eventually type of linker(s)], a compact TALEN
according to the present invention can comprise different levels of
separate enzymatic activities able to differently process DNA,
resulting in a global DNA processing efficiency for said compact
TALEN, each one of said different enzymatic activities having their
own DNA processing efficiency.
[0086] In another preferred embodiment, the method according to the
present invention further comprises the steps of: [0087] (i)
Engineering at least one enhancer domain; [0088] (ii) Optionally
determining or engineering one peptide linker to fuse said enhancer
domain to one part of said compact TALEN entity; thereby obtaining
a compact TALEN entity with enhanced DNA processing efficiency
nearby a single double-stranded DNA target sequence of interest,
i.e. an enhanced compact TALEN.
[0089] In other words, according to the method of the present
invention said unique compact TALEN monomer further comprises:
[0090] (i) At least one enhancer domain; [0091] (ii) Optionally one
peptide linker to fuse said enhancer domain to one part of said
unique compact TALEN monomer active entity.
[0092] In another more preferred embodiment, said enhancer domain
is fused to the N-terminus of the core TALE scaffold part of said
compact TALEN entity. In another more preferred embodiment, said
enhancer domain is fused to C-terminus of the core TALE scaffold
part of said compact TALEN entity. In another more preferred
embodiment, said enhancer domain is fused to the catalytic domain
part of said compact TALEN entity. In another more preferred
embodiment, said enhancer domain is fused between the N-terminus of
the core TALE scaffold part and the catalytic part of said compact
TALEN entity. In another more preferred embodiment, said enhancer
domain is fused between the C-terminus of the core TALE scaffold
part and the catalytic part of said compact TALEN entity. In the
scope of the present invention, it can be envisioned to insert said
catalytic domain and/or enhancer domain between two parts of the
engineered core TALE scaffold according to the invention, each part
comprising one set of RVDs. In this last case, the number of RVDs
for each engineered core TALE scaffold can be the same or not. In
other words, it can be envisioned to split said core TALE scaffold
of the present invention to insert one catalytic domain and/or one
enhancer domain between the resulting two parts of said engineered
core TALE scaffold.
[0093] In another preferred embodiment, said enhancer domain is
catalytically active or not, providing functional and/or structural
support to said compact TALEN entity. In a more preferred
embodiment, said enhancer domain consists of a protein domain
selected from the group consisting of MmeI, Colicin-E7
(CEA7_ECOLX), Colicin-E9, APFL, EndA, Endo I (END1_ECOLI), Human
Endo G (NUCG_HUMAN), Bovine Endo G (NUCG_BOVIN), R.HinP1I, I-BasI,
I-BmoI, I-HmuI, I-TevI, I-TevII, I-TevIII, I-TwoI, R.MspI, R.MvaI,
NucA, NucM, Vvn, Vvn_CLS, Staphylococcal nuclease (NUC_STAAU),
Staphylococcal nuclease (NUC_STAHY), Micrococcal nuclease
(NUC_SHIFL), Endonuclease yncB, Endodeoxyribonuclease I
(ENRN_BPT7), Metnase, Nb.BsrDI, BsrDI A, Nt.BspD6I (R.BspD6I large
subunit), ss.BspD6I (R.BspD6I small subunit), R.PleI, MlyI, AlwI,
Mva1269I, BsrI, BsmI, Nb.BtsCI, Nt.BtsCI, R1.BtsI, R2.BtsI, BbvCI
subunit 1, BbvCI subunit 2, Bpu10I alpha subunit, Bpu10I beta
subunit, BmrI, BfiI, I-CreI, hExol (EXO1_HUMAN), Yeast Exol
(EXO1_YEAST), E. coli Exol, Human TREX2, Mouse TREX1, Human TREX1,
Bovine TREX1, Rat TREX1, Human DNA2, Yeast DNA2 (DNA2_YEAST) and
VP16, as listed in Table 2 (SEQ ID NO: 10 to SEQ ID NO: 66 and SEQ
ID NO: 1, 366 & 367, a functional mutant, a variant or a
derivative thereof. In another more preferred embodiment, said
enhancer domain consists of a catalytically active derivative of
the protein domains listed above and in Table 2, providing
functional and/or structural support to said compact TALEN entity.
In another preferred embodiment, said enhancer domain consists of a
catalytically inactive derivative of the protein domains listed
above and in Table 2, providing structural support to said compact
TALEN entity. In another preferred embodiment, said enhancer domain
is selected from the group consisting of I-TevI (SEQ ID NO: 20),
ColE7 (SEQ ID NO: 11) and NucA (SEQ ID NO: 26).
[0094] In a more preferred embodiment, said enhanced compact TALEN
according to the method of the present invention can comprise a
second enhancer domain. In this embodiment, said second enhancer
domain can have the same characteristics than the first enhancer
domain. In a more preferred embodiment, said second enhancer domain
provides structural support to enhanced compact TALEN entity. In
another more preferred embodiment, said second enhancer domain
provides functional support to enhanced compact TALEN entity. In a
more preferred embodiment, said second enhancer domain provides
structural and functional support to the enhanced compact TALEN
entity. In a more preferred embodiment, said enhanced compact TALEN
entity comprises one catalytic domain and one enhancer domain. In
another more preferred embodiment said enhanced compact TALEN
entity comprises one catalytic domain and two enhancer domains. In
another more preferred embodiment said enhanced compact TALEN
entity comprises two catalytic domains and one enhancer domains. In
another more preferred embodiment said enhanced compact TALEN
entity comprises two catalytic domains and two enhancer
domains.
[0095] In a more preferred embodiment, said second enhancer domain
consists of a protein domain derived from a protein selected from
the group consisting of MmeI, Colicin-E7 (CEA7_ECOLX), Colicin-E9,
APFL, EndA, Endo I (END1_ECOLI), Human Endo G (NUCG_HUMAN), Bovine
Endo G (NUCG_BOVIN), R.HinP1I, I-BasI, I-BmoI, I-HmuI, I-TevI,
I-TevII, I-TevIII, I-TwoI, R.MspI, R.MvaI, NucA, NucM, Vvn,
Vvn_CLS, Staphylococcal nuclease (NUC_STAAU), Staphylococcal
nuclease (NUC_STAHY), Micrococcal nuclease (NUC_SHIFL),
Endonuclease yncB, Endodeoxyribonuclease I (ENRN_BPT7), Metnase,
Nb.BsrDI, BsrDI A, Nt.BspD6I (R.BspD6I large subunit), ss.BspD6I
(R.BspD6I small subunit), R.PleI, MlyI, AiwI, Mva12691, BsrI, BsmI,
Nb.BtsCI, Nt.BtsCI, R1.BtsI, R2.BtsI, BbvCI subunit 1, BbvCI
subunit 2, Bpu10I alpha subunit, Bpu10I beta subunit, BmrI, BfiI,
I-CreI, hExol (EXO1_HUMAN), Yeast Exol (EXO1_YEAST), E. coli Exol,
Human TREX2, Mouse TREX1, Human TREX1, Bovine TREX1, Rat TREX1,
Human DNA2, Yeast DNA2 (DNA2_YEAST) and VP16, as listed in Table 2
(SEQ ID NO: 10 to SEQ ID NO: 66 and SEQ ID NO: 1, 366 & 367, a
functional mutant, a variant or a derivative thereof. In another
more preferred embodiment, said second enhancer domain consists of
a catalytically active derivative of the protein domains listed
above and in Table 2, providing functional and/or structural
support to said enhanced compact TALEN entity. In another preferred
embodiment, said second enhancer domain consists of a catalytically
inactive derivative of the protein domains listed above and in
Table 2, providing structural support to said enhanced compact
TALEN entity.
[0096] In another more preferred embodiment, any combinations of
catalytic and/or enhancer domains listed above, as non-limiting
examples, can be envisioned to be fused to said core TALE scaffold
providing structural and/or functional support to said compact
TALEN entity. More preferably, combinations of catalytic domains
selected from the group of TevI (SEQ ID NO: 20), ColE7 (SEQ ID NO:
11) and NucA (SEQ ID NO: 26) can be envisioned. Optionally, FokI
(SEQ ID NO: 368) can be used in combination with another catalytic
domain according to the list of Tablet. Such combinations of
catalytic and/or enhancer domains can be envisioned regarding the
envisioned applications for using the method of the present
invention.
[0097] Depending from its structural composition [type of core TALE
scaffold, type of catalytic domain(s) with associated enzymatic
activities, eventually type of linker(s) and type of enhancer(s)
domains], an enhanced compact TALEN according to the present
invention can present different levels of separate enzymatic
activities able to differently process DNA, resulting in a global
DNA processing efficiency for said enhanced compact TALEN, each one
of said different enzymatic activities having their own DNA
processing efficiency.
[0098] In this preferred embodiment, the DNA processing efficiency
of the compact TALEN entity according to the method of the present
invention can be enhanced by the engineering of at least one
enhancer domain and one peptidic linker thereby obtaining a compact
TALEN entity with enhanced DNA processing activity nearby a single
double-stranded DNA target sequence of interest, i.e. a enhanced
compact TALEN according to the present invention.
[0099] Depending on its structural composition, the global DNA
processing efficiency that is enhanced in said enhanced compact
TALEN according to the present invention, can have a dominant
enzymatic activity selected from the group consisting of a nuclease
activity, a polymerase activity, a kinase activity, a phosphatase
activity, a methylase activity, a topoisomerase activity, an
integrase activity, a transposase activity or a ligase activity as
non-limiting examples. In a more preferred embodiment, the global
DNA processing efficiency that is enhanced in said enhanced compact
TALEN according to the present invention is a combination of
different enzymatic activities selected from the group consisting
of a nuclease activity, a polymerase activity, a kinase activity, a
phosphatase activity, a methylase activity, a topoisomerase
activity, an integrase activity, a transposase activity or a ligase
activity as non-limiting examples. In a more preferred embodiment,
the global DNA processing efficiency that is enhanced in said
enhanced compact TALEN according to the present invention is one of
its different enzymatic activities selected from the group
consisting of a nuclease activity, a polymerase activity, a kinase
activity, a phosphatase activity, a methylase activity, a
topoisomerase activity, an integrase activity, a transposase
activity or a ligase activity as non-limiting examples. In this
case, the global DNA processing efficiency is equivalent to one DNA
processing activity amongst the enzymatic activities mentioned
above. In another more preferred embodiment, said DNA processing
activity of the compact TALEN entity which is enhanced by the
enhancer is a cleavase activity or a nickase activity or a
combination of both a cleavase activity and a nickase activity.
[0100] Enhancement of DNA processing efficiency of a compact TALEN
entity according to the present invention can be a consequence of a
structural support by at least one enhancer domain. In a preferred
embodiment, said structural support enhances the binding of a
compact TALEN entity according to the invention for said DNA target
sequence compared to the binding of a starting compact TALEN entity
for the same DNA target sequence, thereby indirectly assisting the
catalytic domain(s) to obtain a compact TALEN entity with enhanced
DNA processing activity. In another preferred embodiment, said
structural support enhances the existing catalytical activity of a
compact TALEN entity for a DNA target sequence compared to the
binding of a starting compact TALEN entity for the same DNA target
sequence to obtain a compact TALEN entity with enhanced DNA
processing activity.
[0101] In another preferred embodiment, said enhancer according to
the method of the present invention both enhances the binding of
the compact TALEN entity for said DNA target sequence and the
catalytic activity of the catalytic domain(s) to obtain a compact
TALEN entity with enhanced DNA processing activity. All these
non-limiting examples lead to a compact TALEN entity with enhanced
DNA processing efficiency for a DNA target sequence at a genomic
locus of interest, i.e. an enhanced compact TALEN according to the
present invention.
[0102] Enhancement of DNA processing efficiency of a compact TALEN
entity according to the present invention, compared to a starting
compact TALEN entity, can also be a consequence of a fuctional
support by at least one enhancer domain. In a preferred embodiment,
said functional support can be the consequence of the hydrolysis of
additional phosphodiester bonds. In a more preferred embodiment,
said functional support can be the hydrolysis of additional
phosphodiester bonds by a protein domain derived from a nuclease.
In an embodiment, said functional support can be the hydrolysis of
additional phosphodiester bonds by a protein domain derived from an
endonuclease. In a more preferred embodiment, said functional
support can be the hydrolysis of additional phosphodiester bonds by
a protein domain derived from a cleavase. In another more preferred
embodiment, said functional support can be the hydrolysis of
additional phosphodiester bonds by a protein domain derived from a
nickase. In a more preferred embodiment, said functional support
can be the hydrolysis of additional phosphodiester bonds by a
protein domain derived from an exonuclease.
[0103] In genome engineering experiments, the efficiency of
rare-cutting endonuclease, e.g. their ability to induce a desired
event (Homologous gene targeting, targeted mutagenesis, sequence
removal or excision) at a locus, depends on several parameters,
including the specific activity of the nuclease, probably the
accessibility of the target, and the efficacy and outcome of the
repair pathway(s) resulting in the desired event (homologous repair
for gene targeting, NHEJ pathways for targeted mutagenesis).
[0104] Cleavage by peptidic rare cutting endonucleases usually
generates cohesive ends, with 3' overhangs for LAGLIDADG
meganucleases (Chevalier and Stoddard 2001) and 5' overhangs for
Zinc Finger Nucleases (Smith, Bibikova et al. 2000). These ends,
which result from hydrolysis of phosphodiester bonds, can be
re-ligated in vivo by NHEJ in a seamless way (i.e. a scarless
re-ligation). The restoration of a cleavable target sequence allows
for a new cleavage event by the same endonuclease, and thus, a
series of futile cycles of cleavage and re-ligation events can take
place. Indirect evidences have shown that even in the yeast
Saccharomyces cerevisiae, such cycles could take place upon
continuous cleavage by the HO endonuclease (Lee, Paques et al.
1999). In mammalian cells, several experiment have shown that
perfect re-ligation of compatible cohesive ends resulting from two
independent but close I-SceI-induced DSBs is an efficient process
(Guirouilh-Barbat, Huck et al. 2004; Guirouilh-Barbat, Rass et al.
2007; Bennardo, Cheng et al. 2008; Bennardo, Gunn et al. 2009).
Absence of the Ku DNA repair protein does not significantly affect
the overall frequency of NHEJ events rejoining the ends from the
two DSBs; however it very strongly enhances the contribution of
imprecise NHEJ to the repair process in CHO immortalized cells and
mouse ES cells (Guirouilh-Barbat, Huck et al. 2004;
Guirouilh-Barbat, Rass et al. 2007; Bennardo, Cheng et al. 2008).
Furthermore, the absence of Ku stimulates I-SceI-induced events
such as imprecise NHEJ (Bennardo, Cheng et al. 2008), single-strand
annealing (Bennardo, Cheng et al. 2008) and gene conversion
(Pierce, Hu et al. 2001; Bennardo, Cheng et al. 2008) in mouse ES
cells. Similar observations shave been made with cells deficient
for the XRCC4 repair protein (Pierce, Hu et al. 2001;
Guirouilh-Barbat, Rass et al. 2007; Bennardo, Gunn et al. 2009)
(although XRCC4 deficiency affects the overal level of NHEJ in CHO
cells (Guirouilh-Barbat, Rass et al. 2007)) or for DNA-PK (Pierce,
Hu et al. 2001). In contrast, knock-down of CtIP has been shown to
suppresses "alt-NHEJ" (a Ku- and XRCC4-independent form of NHEJ
more prone to result in imprecise NHEJ), single-strand annealing
and gene conversion, while not affecting the overall level of
rejoining of two compatible ends generated by I-SceI (Bennardo,
Cheng et al. 2008). Thus, competition between different DSB repair
pathways can affect the spectrum or repair events resulting from a
nuclease-induced DSB.
[0105] In addition, DSB resection is important for certain DSB
pathways. Extensive DSB resection, resulting in the generation of
large single stranded regions (a few hundred nucleotides at least),
has been shown in yeast to initiate single strand annealing
(Sugawara and Haber 1992) and strand invasion, the ATP-dependant
step that initiates many homologous recombination events of DNA
duplex invasion by an homologous strand that (White and Haber 1990;
Sun, Treco et al. 1991) (for a review of mechanisms, see (Paques
and Haber 1999)). In eukaryotic cells DSB resection depends on
several proteins including BLM/Sgs1 and DNA2, EXOI, and the MRN
complex (Mre11, Rad50, Nbs1/Xrs2) and is thought to result from
different pathways. MRN is involved in a small scale resection
process, while two redundant pathways depending on BLM and DNA2 on
one hand, and on EXOI on another hand, would be involved in
extensive resection (Mimitou and Symington 2008; Nimonkar, Genschel
et al. 2011). In addition, processing ends involving a damaged
nucleotide (resulting from chemical cleavage or from a bulk
adduct), requires the CtlP/Sae2 protein together with RMN (Sartori,
Lukas et al. 2007; Buis, Wu et al. 2008; Hartsuiker, Mizuno et al.
2009). Over-expression of the Trex2 exonuclease was shown to
strongly stimulate imperfect NHEJ associated with loss of only a
few base pairs (Bennardo, Gunn et al. 2009), while it inhibited
various kinds of DNA repair events between distant sequences (such
as Single-strand annealing, NHEJ between ends from different
breaks, or NHEJ repair of a single DSB involving remote
micro-homologies). In the same study, it was suggested that Trex2
did resect the 3' overhangs let by I-SceI in a non processive way.
Thus, the type of stimulated pathway could in turn depend on the
type of resection (length of resection, single strand vs. double
strand, resection of 5' strand vs. 3' strand).
[0106] Thus, the efficiency of a compact TALEN, e.g. it ability to
produce a desired event such as targeted mutagenesis or homologous
gene targeting (see definition for full definition of "efficiency
of compact TALEN"), can be enhanced by an enhancement or
modification of its global DNA processing efficiency (see
definition for full definition of "global DNA processing
efficiency"), e.g. the global resultant or the overall result of
different separate enzymatic activities that said compact
TALEN.
[0107] In a preferred embodiment, enhancement of global DNA
processing efficiency of a compact TALEN entity according to the
present invention, compared to a starting compact TALEN entity, can
be the hydrolysis of additional phosphodiester bonds at the
cleavage site.
[0108] Said hydrolysis of additional phosphodiester bonds at the
cleavage site by said at least one enhancer according to the
invention can lead to different types of DSB resection affecting at
said DSB cleavage site, one single DNA strand or both DNA strands,
affecting either 5' overhangs ends, either 3' overhangs ends, or
both ends and depending on the length of said resection. Thus,
adding new nickase or cleavase activities to the existing cleavase
activity of a compact TALEN entity can enhance the efficiency of
the resulting enhanced compact TALEN according to the invention, at
a genomic locus of interest (FIG. 8B-8E). As a non-limiting
example, addition of two nickase activities on opposite strands
(FIG. 8D) or of a new cleavase activity generating a second DSB
(FIG. 8E) can result in a double-strand gap. As a consequence,
perfect religation is no longer possible, and one or several
alternative repair outcomes such as imprecise NHEJ, Homologous
Recombination or SSA for instance, can be stimulated. As another
non-limiting example, the addition of a single nickase activity can
result in a single strand gap, and suppress the cohesiveness of the
ends, which can also enhance the efficiency of the resulting
enhanced compact TALEN at a genomic locus of interest, according to
the invention, via stimulation of one or several alternative repair
outcomes mentioned above.
[0109] In this aspect of the present invention, enhancement of DNA
processing efficiency of a compact TALEN refers to the increase in
the detected level of said DNA processing efficiency, against a
target DNA sequence, of a enhanced compact TALEN in comparison to
the activity of a first compact TALEN against the same target DNA
sequence. Said first compact TALEN can be a starting compact TALEN,
or a compact TALEN that has already been engineered or an enhanced
compact TALEN according to the present invention. Several rounds of
enhancement can be envisioned from a starting compact TALEN or from
a starting enhanced compact TALEN.
[0110] In this aspect of the method of the present invention,
enhancement of the DNA processing efficiency of the compact TALEN
entity (or enhanced compact TALEN) refers to the increase in the
detected level of said DNA processing efficiency against a target
DNA sequence of interest or nearby said DNA sequence of interest in
comparison to the efficiency of a first compact TALEN or starting
compact TALEN against or nearby the same target DNA sequence. In
this case, the starting compact TALEN is taken as the reference
scaffold to measure the DNA processing efficiency. Said enhanced
compact TALEN is an engineered compact TALEN comprising an enhancer
domain according to this aspect of the invention. Said enhanced
compact TALEN can also be taken as a reference scaffold for further
enhancement of said DNA processing efficiency. As a non-limiting
example, said DNA processing efficiency can result from a
cleavage-induced recombination generated by said enhanced compact
TALEN. In this case, said level of cleavage-induced recombination
can be determined, for instance, by a cell-based recombination
assay as described in the International PCT Application WO
2004/067736. Importantly, enhancement of efficacy in cells
(enhanced generation of targeted mutagenesis or targeted
recombination) can be, but is not necessarily associated with an
enhancement of the cleavage activity that could be detected in
certain in vitro assays. For example, additional phosphodiesterase
activities as described in FIG. 8 could barely affect the cleavage
profile, as detected by in vitro cleavage and separation of the
cleavage products on an electrophoresis gel. However, as explained
above, and in the legend of FIG. 8, the DSB ends generated in this
way could be more prone to induce detectable genomic rearrangements
such as targeted mutagenesis (by imprecise NHEJ) or homologous
recombination. Said enhancement in cleavage-induced recombination
of said enhanced compact TALEN is at least a 5% enhancement
compared to the starting scaffold or starting compact TALEN, more
preferably at least a 10% enhancement, again more preferably at
least a 15% enhancement, again more preferably at least a 20%
enhancement, again more preferably at least a 25% enhancement,
again more preferably a 50% enhancement, again more preferably an
enhancement greater than 50%, resulting in an enhancement of DNA
processing efficiency of said enhanced compact TALEN of at least 5%
compared to the starting scaffold or starting compact TALEN, more
preferably at least a 10% enhancement, again more preferably at
least a 15% enhancement, again more preferably at least a 20%
enhancement, again more preferably at least a 25% enhancement,
again more preferably a 50% enhancement, again more preferably a
enhancement greater than 50%.
[0111] In another preferred embodiment according to the method of
the present invention, the peptidic linker that can link said
enhancer domain to one part of said compact TALEN entity according
to the method of the present invention can be selected from the
group consisting of NFS1, NFS2, CFS1, RM2, BQY, QGPSG, LGPDGRKA,
1a8h.sub.--1, 1dnpA.sub.--1, 1d8cA.sub.--2, 1ckqA.sub.--3,
1sbp.sub.--1, 1ev7A.sub.--1, 1alo.sub.--3, 1amf.sub.--1,
1adjA.sub.--3, 1fcdC.sub.--1, 1a13.sub.--2, 1g3p.sub.--1,
1acc.sub.--3, 1ahjB.sub.--1, 1acc.sub.--1, 1af7.sub.--1,
1heiA.sub.--1, 1bia.sub.--2, 1igtB.sub.--1, 1nfkA.sub.--1,
1au7A.sub.--1, 1bpoB.sub.--1, 1b0pA.sub.--2, 1c05A.sub.--2,
1gcb.sub.--1, 1bt3A.sub.--1, 1b3o13.sub.--2, 16vpA.sub.--6,
1dhx.sub.--1, 1b8aA.sub.--1 and 1qu6A.sub.--1 as listed in Table 3
(SEQ ID NO: 67 to SEQ ID NO: 104 and SEQ ID NO: 372 to SEQ ID NO:
415). In a more preferred embodiment, the peptidic linker that can
said enhancer domain to one part of said compact TALEN entity
according to the method of the present invention can be selected
from the group consisting of NFS1 (SEQ ID NO: 98), NFS2 (SEQ ID NO:
99) and CFS1 (SEQ ID NO: 100). In the scope of the present
invention is also encompassed the case where a peptidic linker is
not needed to fuse one enhancer domain to one part of said compact
TALEN entity in order to obtain a enhanced compact TALEN according
to the present invention.
[0112] Depending from its structural composition [type of core TALE
scaffold, type of catalytic domain(s) with associated enzymatic
activities, type of enhancers and eventually type of linker(s)], a
compact TALEN or an enhanced compact TALEN according to the present
invention can comprise different levels of separate enzymatic
activities able to differently process DNA as mentioned above. By
adding new enzymatic activities to said compact TALEN or said
enhanced compact TALEN or enhancing the DNA processing efficiency
of one or several of its constitutive enzymatic activities, one can
enhance the global DNA processing efficiency of one compact TALEN
or enhanced compact TALEN in comparison to a starting compact TALEN
or enhanced compact TALEN.
[0113] According to the present invention, compact TALENs are
designed to alleviate the need for multiple independent protein
moieties when targeting a DNA processing event. Importantly, the
requisite "spacer" region and dual target sites essential for the
function of current TALENs are unnecessary. As each end of the core
TALE scaffold is amenable to fusion, the order (N- v.s C-terminal)
of addition of the catalytic and enhancement domains can vary with
the application. In addition, since the catalytic domain does not
require specific DNA contacts, there are no restrictions on regions
surrounding the core TALE scaffold, as non-limiting examples
depicted in FIG. 5: (A) N-terminal fusion construct to promote
Homologous recombination induced by a cleavase domain or by a
nickase domain. (B) C-terminal fusion construct with properties as
in (A). (C) The attachment of two catalytic domains to both ends of
the core TALE scaffold allows for dual cleavage with enhancement in
NHEJ. Fusion junctions (N-vs. C-terminal) and linker designs can
vary with the application.
[0114] According to the present invention, compact TALENs can be
enhanced through the addition of a domain to promote existing or
alternate activities as non-limiting examples depicted in FIG. 6:
(A) A standard compact TALEN with an enhancer domain fused to the
C-terminus of its core TALE scaffold part. (B) The enhancer domain
is fused to the compact TALEN via the N-terminus of its catalytic
domain part. Such a configuration can be used to assist and/or
anchor the catalytic domain part near the DNA to increase DNA
processing activity. (C) The enhancer domain is sandwiched between
the catalytic domain part and the core TALE scaffold part. The
enhancer domain can promote communication between the flanking
domains (i.e. to assist in catalysis and/or DNA binding) or can be
used to overcome the requisite T nucleotide at position -1 of all
TALE-based targets. (D) The enhancer domain is used to functionally
replace the engineered core TALE scaffold N-terminal region. (E)
The enhancer domain is used to functionally replace the engineered
core TALE scaffold C-terminal region. Fusion junctions (N-vs.
C-terminal) and linker designs can vary with the application.
[0115] According to the present invention, the nature of the
catalytic domain(s) comprised in the compact TALEN and the enhanced
compact TALEN is application dependent. As a non-limiting example,
a nickase domain should allow for a higher HR/NHEJ ratio than a
cleavase domain, thereby being more agreeable for therapeutic
applications (McConnell Smith, Takeuchi et al. 2009; Metzger,
McConnell-Smith et al. 2011). For example, the coupling of a
cleavase domain on one side with a nickase domain on the other
could result in excision of a single-strand of DNA spanning the
binding region of a compact TALEN. The targeted generation of
extended single-strand overhangs could be applied in applications
that target DNA repair mechanisms. For targeted gene inactivation,
the use of two cleavase domains is then preferred. In another
preferred embodiment, the use of two nickase domains can be
favored. Furthermore, the invention relates to a method for
generating several distinct types of compact TALENs that can be
applied to applications ranging from targeted DNA cleavage to
targeted gene regulation.
[0116] In another aspect, the present invention relates to a
compact TALEN comprising: [0117] (i) One core TALE scaffold
comprising different sets of Repeat Variable Dipeptide regions
(RVDs) to change DNA binding specificity and target a specific
single double-stranded DNA target sequence of interest, onto which
a selection of catalytic domains can be attached to effect DNA
processing; [0118] (ii) At least one catalytic domain wherein said
catalytic domain is capable of processing DNA nearby said single
double-stranded DNA target sequence of interest when fused to said
engineered core TALE scaffold from (i); [0119] (iii) Optionally one
peptidic linker to fuse said catalytic domain from (ii) to said
engineered core TALE scaffold from (i) when needed; such that said
compact TALEN does not require dimerization to target a specific
single double-stranded DNA target sequence of interest and process
DNA nearby said single double-stranded DNA target sequence of
interest. In other words, the compact TALEN according to the
present invention is an active entity unit able, by itself, to
target only one specific single double-stranded DNA target sequence
of interest through one DNA binding domain and to process DNA
nearby said single double-stranded DNA target sequence of
interest.
[0120] The present invention relates to a compact TALEN monomer
comprising: [0121] (i) One core TALE scaffold comprising Repeat
Variable Dipeptide regions (RVDs) having DNA binding specificity
onto a specific double-stranded DNA target sequence of interest;
[0122] (ii) At least one catalytic domain wherein said catalytic
domain is capable of processing DNA a few base pairs away from said
double-stranded DNA target sequence of interest when fused to the C
or N terminal of said core TALE scaffold from (i); [0123] (iii)
Optionally one peptidic linker to fuse said catalytic domain from
(ii) to said engineered core TALE scaffold from (i) when needed;
wherein said compact TALEN monomer is assembled to bind said target
DNA sequence and process double-stranded DNA without requiring
dimerization.
[0124] In another embodiment, said engineered core TALE scaffold of
the compact TALEN according to the present invention comprises an
additional N-terminal domain resulting in an engineered core TALE
scaffold sequentially comprising a N-terminal domain and different
sets of Repeat Variable Dipeptide regions (RVDs) to change DNA
binding specificity and target a specific single double-stranded
DNA target sequence of interest, onto which a selection of
catalytic domains can be attached to effect DNA processing.
[0125] In another embodiment, said engineered core TALE scaffold of
the compact TALEN according to the present invention comprises an
additional C-terminal domain resulting in an engineered core TALE
scaffold sequentially comprising different sets of Repeat Variable
Dipeptide regions (RVDs) to change DNA binding specificity and
target a specific single double-stranded DNA target sequence of
interest and a C-terminal domain, onto which a selection of
catalytic domains can be attached to effect DNA processing.
[0126] In another embodiment, said engineered core TALE-scaffold of
the compact TALEN according to the present invention comprises
additional N-terminus and a C-terminal domains resulting in an
engineered core TALE scaffold sequentially comprising a N-terminal
domain, different sets of Repeat Variable Dipeptide regions (RVDs)
to change DNA binding specificity and target a specific single
double-stranded DNA target sequence of interest and a C-terminal
domain, onto which a selection of catalytic domains can be attached
to effect DNA processing.
[0127] In another embodiment, said engineered core TALE-scaffold
according to the present invention comprises the protein sequences
selected from the group consisting of ST1 (SEQ ID NO: 134) and ST2
(SEQ ID NO: 135). In another embodiment, said engineered core TALE
scaffold comprises a protein sequence having at least 80%, more
preferably 90%, again more preferably 95% amino acid sequence
identity with the protein sequences selected from the group
consisting of SEQ ID NO: 134 and SEQ ID NO: 135. In another
embodiment, said engineered core TALE-scaffold according to the
present invention comprises the protein sequences selected from the
group consisting of bT1-Avr (SEQ ID NO: 136), bT2-Avr (SEQ ID NO:
137), bT1-Pth (SEQ ID NO: 138) and bT2-Pth (SEQ ID NO: 139). In
another embodiment, said engineered TALE-scaffold comprises a
protein sequence having at least 80%, more preferably 90%, again
more preferably 95% amino acid sequence identity with the protein
sequences selected from the group consisting of SEQ ID NO: 136 to
SEQ ID NO: 139.
[0128] In a preferred embodiment, said additional N-terminus and
C-terminal domains of engineered core TALE scaffold are derived
from natural TALE. In a more preferred embodiment said additional
N-terminus and C-terminal domains of engineered core TALE scaffold
are derived from natural TALE selected from the group consisting of
AvrBs3, PthXo1, AvrHah1, PthA, Tal1c as non-limiting examples. In
another more preferred embodiment, said additional N-terminus
and/or said C-terminal domains are truncated forms of respective
N-terminus and/or said C-terminal domains of natural TALE like
AvrBs3, PthXo1, AvrHah1, PthA, Tal1c as non-limiting examples, from
which they are derived. In a more preferred embodiment, said
additional N-terminus and C-terminal domains sequences of
engineered core TALE scaffold are selected from the group
consisting of ST1 SEQ ID NO: 134 and ST2 SEQ ID NO: 135 as
respectively exemplified in baseline protein scaffolds bT1-Avr (SEQ
ID NO: 136) or bT1-Pth (SEQ ID NO: 138) and bT2-Avr (SEQ ID NO:
137) or bT2-Pth (SEQ ID NO: 139).
[0129] In another embodiment, each RVD of said core scaffold is
made of 30 to 42 amino acids, more preferably 33 or 34 wherein two
critical amino acids located at positions 12 and 13 mediates the
recognition of one nucleotide of said nucleic acid target sequence;
equivalent two critical amino acids can be located at positions
other than 12 and 13 specialy in RVDs taller than 33 or 34 amino
acids long. Preferably, RVDs associated with recognition of the
different nucleotides are HD for recognizing C, NG for recognizing
T, NI for recognizing A, NN for recognizing G or A, NS for
recognizing A, C, G or T, HG for recognizing T, IG for recognizing
T, NK for recognizing G, HA for recognizing C, ND for recognizing
C, HI for recognizing C, HN for recognizing G, NA for recognizing
G, SN for recognizing G or A and YG for recognizing T, TL for
recognizing A, VT for recognizing A or G and SW for recognizing A.
More preferably, RVDs associated with recognition of the
nucleotides C, T, A, G/A and G respectively are selected from the
group consisting of NN or NK for recognizing G, HD for recognizing
C, NG for recognizing T and NI for recognizing A, TL for
recognizing A, VT for recognizing A or G and SW for recognizing A.
In another embodiment, RVDS associated with recognition of the
nucleotide C are selected from the group consisting of N* and RVDS
associated with recognition of the nucleotide T are selected from
the group consisting of N* and H*, where * denotes a gap in the
repeat sequence that corresponds to a lack of amino acid residue at
the second position of the RVD. In another embodiment, critical
amino acids 12 and 13 can be mutated towards other amino acid
residues in order to modulate their specificity towards nucleotides
A, T, C and G and in particular to enhance this specificity. By
other amino acid residues is intended any of the twenty natural
amino acid residues or unnatural amino acids derivatives.
[0130] In another embodiment, said core scaffold of the present
invention comprises between 8 and 30 RVDs. More preferably, said
core scaffold of the present invention comprises between 8 and 20
RVDs; again more preferably 15 RVDs.
[0131] In another embodiment, said core scaffold comprises an
additional single truncated RVD made of 20 amino acids located at
the C-terminus of said set of RVDs, i.e. an additional C-terminal
half-RVD. In this case, said core scaffold of the present invention
comprises between 8.5 and 30.5 RVDs, "0.5" referring to previously
mentioned half-RVD (or terminal RVD, or half-repeat). More
preferably, said core scaffold of the present invention comprises
between 8.5 and 20.5 RVDs, again more preferably, 15.5 RVDs. In a
preferred embodiment, said half-RVD is in a core scaffold context
which allows a lack of specificity of said half-RVD toward
nucleotides A, C, G, T. In a more preferred embodiment, said
half-RVD is absent.
[0132] In another embodiment, said core scaffold of the present
invention comprises RVDs of different origins. In a preferred
embodiment, said core scaffold comprises RVDs originating from
different naturally occurring TAL effectors. In another preferred
embodiment, internal structure of some RVDs of the core scaffold of
the present invention are constituted by structures or sequences
originated from different naturally occurring TAL effectors. In
another embodiment, said core scaffold of the present invention
comprises RVDs-like domains. RVDs-like domains have a sequence
different from naturally occurring RVDs but have the same function
and/or global structure within said core scaffold of the present
invention.
[0133] In another embodiment, said additional N-terminal domain of
said engineered core TALE scaffold of said compact TALEN according
to the present invention is an enhancer domain. In another
embodiment, said enhancer domain is selected from the group
consisting of Puf RNA binding protein or Ankyrin super-family, as
non-limiting examples. In another embodiment, said enhancer domain
sequence is selected from the group consisting of protein domains
of SEQ ID NO: 4 and SEQ ID NO: 5 as non-limiting examples listed in
Table 1, a functional mutant, a variant or a derivative thereof. In
another embodiment, said additional C-terminal domain of said
engineered core TALE scaffold is an enhancer domain. In another
embodiment, said enhancer domain is selected from the group
consisting of hydrolase/transferase of Pseudomonas Aeuriginosa
family, the polymerase domain from the Mycobacterium tuberculosis
Ligase D family, the initiation factor elF2 from Pyrococcus family,
the translation initiation factor Aif2 family as non-limiting
examples. In another embodiment, said enhancer domain sequence is
selected from the group consisting of protein domains of SEQ ID NO:
6 to SEQ ID NO: 9 as non-limiting examples listed in Table 1.
[0134] In another preferred embodiment, the catalytic domain that
is capable of processing DNA nearby the single double-stranded DNA
target sequence of interest, when fused to said engineered core
TALE scaffold according to the present invention, is fused to the
N-terminus part of said core TALE scaffold. In another preferred
embodiment, said catalytic domain is fused to the C-terminus part
of said core TALE scaffold. In another preferred embodiment two
catalytic domains are fused to both N-terminus part of said core
TALE scaffold and C-terminus part of said core TALE scaffold. In a
more preferred embodiment, said catalytic domain has an enzymatic
activity selected from the group consisting of nuclease activity,
polymerase activity, kinase activity, phosphatase activity,
methylase activity, topoisomerase activity, integrase activity,
transposase activity or ligase activity. In another preferred
embodiment, the catalytic domain fused to the core TALE scaffold of
the present invention can be a transcription activator or repressor
(i.e. a transcription regulator), or a protein that interacts with
or modifies other proteins such as histones. Non-limiting examples
of DNA processing activities of said compact TALEN of the present
invention include, for example, creating or modifying epigenetic
regulatory elements, making site-specific insertions, deletions, or
repairs in DNA, controlling gene expression, and modifying
chromatin structure.
[0135] In another more preferred embodiment, said catalytic domain
has an endonuclease activity. In another more preferred embodiment,
said catalytic domain of the compact TALEN according to the present
invention has cleavage activity on said double-stranded DNA
according to the method of the present invention. In another more
preferred embodiment, said catalytic domain has a nickase activity
on said double-stranded DNA according to the method of the present
invention. In another more preferred embodiment, said catalytic
domain is selected from the group consisting of proteins MmeI,
Colicin-E7 (CEA7_ECOLX), Colicin-E9, APFL, EndA, Endo I
(END1_ECOLI), Human Endo G (NUCG_HUMAN), Bovine Endo G
(NUCG_BOVIN), R.HinP1I, I-BasI, I-BmoI, I-HmuI, I-TevI, I-TevII,
I-TevIII, I-TwoI, R.MspI, R.MvaI, NucA, NucM, Vvn, Vvn_CLS,
Staphylococcal nuclease (NUC_STAAU), Staphylococcal nuclease
(NUC_STAHY), Micrococcal nuclease (NUC_SHIFL), Endonuclease yncB,
Endodeoxyribonuclease I (ENRN_BPT7), Metnase, Nb.BsrDI, BsrDI A,
Nt.BspD6I (R.BspD6I large subunit), ss.BspD6I (R.BspD6I small
subunit), R.PleI, MlyI, AlwI, Mva1269I, BsrI, BsmI, Nb.BtsCI,
Nt.BtsCI, R1.BtsI, R2.BtsI, BbvCI subunit 1, BbvCI subunit 2,
Bpu10I alpha subunit, Bpu10I beta subunit, BmrI, BfiI, I-CreI,
hExol (EXO1_HUMAN), Yeast Exol (EXO1_YEAST), E. coli Exol, Human
TREX2, Mouse TREX1, Human TREX1, Bovine TREX1, Rat TREX1, Human
DNA2, Yeast DNA2 (DNA2_YEAST) and VP16, as listed in Table 2 (SEQ
ID NO: 10 to SEQ ID NO: 66 and SEQ ID NO: 1, 366 & 367), a
functional mutant, a variant or a derivative thereof. In another
preferred embodiment said catalytic domain of the compact TALEN
according to the present invention is I-TevI (SEQ ID NO: 20), a
functional mutant, a variant or a derivative thereof. In another
preferred embodiment, catalytic domain I-TevI (SEQ ID NO: 20), a
functional mutant, a variant or a derivative thereof is fused to
the N-terminal domain of said core TALE scaffold according to the
compact TALEN of the present invention. In another preferred
embodiment, said compact TALEN according to the present invention
comprises a protein sequence having at least 80%, more preferably
90%, again more preferably 95% amino acid sequence identity with
the protein sequences selected from the group of SEQ ID NO:
426-432.
[0136] In another preferred embodiment, said catalytic domain of
the compact TALEN according to the present invention is ColE7 (SEQ
ID NO: 11), a functional mutant, a variant or a derivative thereof.
In another preferred embodiment, catalytic domain ColE7 (SEQ ID NO:
11), a functional mutant, a variant or a derivative thereof is
fused to the N-terminal domain of said core TALE scaffold according
to the method of the present invention. In another preferred
embodiment, catalytic domain ColE7 (SEQ ID NO: 11), a functional
mutant, a variant or a derivative thereof is fused to the
C-terminal domain of said core TALE scaffold according to the
method of the present invention. In another preferred embodiment,
said compact TALEN according to the method of the present invention
comprises a protein sequence having at least 80%, more preferably
90%, again more preferably 95% amino acid sequence identity with
the protein sequences selected from the group of SEQ ID NO:
435-438.
[0137] In another preferred embodiment, said catalytic domain of
the compact TALEN according to the present invention is NucA (SEQ
ID NO: 26), a functional mutant, a variant or a derivative thereof.
In another preferred embodiment, catalytic domain NucA (SEQ ID NO:
26), a functional mutant, a variant or a derivative thereof is
fused to the N-terminal domain of said core TALE scaffold according
to the method of the present invention. In another preferred
embodiment, catalytic domain NucA (SEQ ID NO: 26), a functional
mutant, a variant or a derivative thereof is fused to the
C-terminal domain of said core TALE scaffold according to the
method of the present invention. In another preferred embodiment,
said compact TALEN according to the method of the present invention
comprises a protein sequence having at least 80%, more preferably
90%, again more preferably 95% amino acid sequence identity with
the protein sequences selected from the group of SEQ ID NO:
433-434.
[0138] In another preferred embodiment, said catalytic domain is
I-CreI (SEQ ID NO: 1), a functional mutant, a variant or a
derivative thereof. In another preferred embodiment, catalytic
domain I-CreI (SEQ ID NO: 1), a functional mutant, a variant or a
derivative thereof is fused to the N-terminal domain of said core
TALE scaffold according to the method of the present invention. In
another preferred embodiment, catalytic domain I-CreI (SEQ ID NO:
1), a functional mutant, a variant or a derivative thereof is fused
to the C-terminal domain of said core TALE scaffold according to
the present invention. In another preferred embodiment, said
compact TALEN according to the present invention comprises a
protein sequence having at least 80%, more preferably 90%, again
more preferably 95% amino acid sequence identity with the protein
sequences selected from the group of SEQ ID NO: 439-441 and SEQ ID
NO: 444-446.
[0139] In another embodiment, said catalytic domain is a
restriction enzyme such as MmeI, R-HinPll, R.MspI, R.MvaI,
Nb.BsrDI, BsrDI A, Nt.BspD6I, ss.BspD6I, R.PleI, MlyI and AlwI as
non-limiting examples listed in table 2. In another more preferred
embodiment, said catalytic domain has an exonuclease activity. In
another more preferred embodiment, any combinations of two
catalytic domains selected from the group consisting of proteins
MmeI, Colicin-E7 (CEA7_ECOLX), Colicin-E9, APFL, EndA, Endo I
(END1_ECOLI), Human Endo G (NUCG_HUMAN), Bovine Endo G
(NUCG_BOVIN), R.HinP1I, I-BasI, I-BmoI, I-HmuI, I-TevI, I-TevII,
I-TevIII, I-TwoI, R.MspI, R.MvaI, NucA, NucM, Vvn, Vvn_CLS,
Staphylococcal nuclease (NUC_STAAU), Staphylococcal nuclease
(NUC_STAHY), Micrococcal nuclease (NUC_SHIFL), Endonuclease yncB,
Endodeoxyribonuclease I (ENRN_BPT7), Metnase, Nb.BsrDI, BsrDI A,
Nt.BspD6I (R.BspD6I large subunit), ss.BspD6I (R.BspD6I small
subunit), R.PleI, MlyI, AlwI, Mva1269I, BsrI, BsmI, Nb.BtsCI,
Nt.BtsCI, R1.BtsI, R2.BtsI, BbvCI subunit 1, BbvCI subunit 2,
Bpu10I alpha subunit, Bpu10I beta subunit, BmrI, BfiI, I-CreI,
hExol (EXO1_HUMAN), Yeast Exol (EXO1_YEAST), E. coli Exol, Human
TREX2, Mouse TREX1, Human TREX1, Bovine TREX1, Rat TREX1, Human
DNA2, Yeast DNA2 (DNA2_YEAST) and VP16, as listed in Table 2 (SEQ
ID NO: 10 to SEQ ID NO: 66 and SEQ ID NO: 1, 366 & 367), a
functional mutant, a variant or a derivative of these protein
domains thereof, can be fused to both N-terminus part and
C-terminus part of said core TALE scaffold, respectively. For
example, I-HmuI catalytic domain can be fused to the N-terminus
part of said core TALE scaffold and ColE7 catalytic domain can be
fused to the C-terminus part of said core TALE scaffold. In another
example, I-TevI catalytic domain can be fused to the N-terminus
part of said core TALE scaffold and ColE7 catalytic domain can be
fused to the C-terminus part of said core TALE scaffold.
[0140] Table 14 below gives non-limiting examples of combinations
of catalytic domains that can be comprised in the compact TALEN
monomer according to the present invention. Optionally, FokI (SEQ
ID NO:368) can be used in combination with another catalytic domain
according to the list of Table2.
TABLE-US-00004 TABLE 14 Examples of combinations of catalytic
domains respectively fused to N and C-terminus part of compact
TALEN core scaffolds according to the present invention leading to
dual-cleavage TALENs. Catalytic domain Catalytic domain fused to
N-terminus fused to C-terminus part of core TALE part of core TALE
Dual-cleavage scaffold scaffold TALENS I-TevI I-TevI TevI-TevI
ColE7 ColE7 ColE7-ColE7 NucA NucA NucA-NucA I-TevI ColE7 TevI-ColE7
I-TevI NucA TevI-NucA ColE7 I-TevI ColE7-TevI ColE7 NucA ColE7-NucA
NucA I-TevI NucA-TevI NucA ColE7 NucA-ColE7
[0141] In a preferred embodiment according to the present
invention, said unique compact TALEN monomer comprises a
combination of two catalytic domains respectively fused to the
N-terminus part and to the C-terminus part of said core TALE
scaffold selected from the group consisting of: [0142] (i) A Nuc A
domain (SEQ ID NO: 26) in N-terminus and a Nuc A domain (SEQ ID NO:
26) in C-terminus; [0143] (ii) A ColE7 domain (SEQ ID NO: 11) in
N-terminus and a ColE7 domain (SEQ ID NO: 11) in C-terminus; [0144]
(iii) A TevI domain (SEQ ID NO: 20) in N-terminus and a ColE7
domain (SEQ ID NO: 11) in C-terminus; [0145] (iv) A TevI domain
(SEQ ID NO: 20) in N-terminus and a NucA domain (SEQ ID NO: 26) in
C-terminus; [0146] (v) A ColE7 domain (SEQ ID NO: 11) in N-terminus
and a NucA domain (SEQ ID NO: 26) in C-terminus; [0147] (vi) A NucA
domain (SEQ ID NO: 26) in N-terminus and a ColE7 domain (SEQ ID NO:
11) in C-terminus.
[0148] In another preferred embodiment, said compact TALEN
according to the present invention comprises a protein sequence
having at least 80%, more preferably 90%, again more preferably 95%
amino acid sequence identity with the protein sequences selected
from the group consisting of SEQ ID NO: 448 and 450.
[0149] In another preferred embodiment, said compact TALEN
according to the present invention comprises a combination of two
catalytic domains respectively fused to the C-terminus part and to
the N-terminus part of said core TALE scaffold selected from the
group consisting of: [0150] (i) A TevI domain (SEQ ID NO: 20) in
N-terminus and a FokI domain (SEQ ID NO: 368) in C-terminus; [0151]
(ii) A TevI domain (SEQ ID NO: 20) in N-terminus and a TevI domain
(SEQ ID NO: 20) in C-terminus; [0152] (iii) A scTrex2 domain (SEQ
ID NO: 451) in N-terminus and a FokI domain (SEQ ID NO: 368) in
C-terminus.
[0153] In another preferred embodiment, said compact TALEN
according to the present invention comprises a protein sequence
having at least 80%, more preferably 90%, again more preferably 95%
amino acid sequence identity with the protein sequences selected
from the group consisting of SEQ ID NO: 447-450 and SEQ ID NO:
452.
[0154] In the scope of the present invention, it can be envisioned
to insert said catalytic domain and/or said enhancer domain between
two parts of the engineered core TALE scaffold according to the
invention, each part comprising one set of RVDs. In this last case,
the number of RVDs for each part of the engineered core TALE
scaffold can be the same or not. In other words, it can be
envisioned to split said core TALE scaffold of the present
invention to insert one catalytic domain and/or one enhancer domain
between the resulting two parts of said engineered core TALE
scaffold. In another preferred embodiment, said compact TALEN
according to the present invention comprises a protein sequence
having at least 80%, more preferably 90%, again more preferably 95%
amino acid sequence identity with the protein sequences selected
from the group consisting of SEQ ID NO: 453-455.
[0155] In other words, the compact TALEN monomer of the present
invention comprises a protein sequence having at least 80%, more
preferably 90%, again more preferably 95% amino acid sequence
identity with the protein sequences selected from the group
consisting of SEQ ID NO: 420-450 and 452-455.
[0156] In another preferred embodiment according to the method of
the present invention, the peptidic linker that can link said
catalytic domain to the core TALE scaffold according to the method
of the present invention can be selected from the group consisting
of NFS1, NFS2, CFS1, RM2, BOY, QGPSG, LGPDGRKA, 1a8h.sub.--1,
1dnpA.sub.--1, 1d8cA.sub.--2, 1ckqA.sub.--3, 1sbp.sub.--1,
1ev7A.sub.--1, 1alo.sub.--3, 1amf 1, 1adjA.sub.--3, 1fcdC.sub.--1,
1a13.sub.--2, 1g3p.sub.--1, 1acc.sub.--3, 1ahjB.sub.--1,
1acc.sub.--1, 1af7.sub.--1, 1heiA.sub.--1, 1bia.sub.--2,
1igtB.sub.--1, 1nfkA.sub.--1, 1au7A.sub.--1, 1 bpoB.sub.--1,
1b0pA.sub.--2, 1c05A.sub.--2, 1gcb.sub.--1, 1bt3A.sub.--1,
1b3o13.sub.--2, 16vpA.sub.--6, 1dhx.sub.--1, 1b8aA.sub.--1 and
1qu6A.sub.--1, as listed in Table 3 (SEQ ID NO: 67 to SEQ ID NO:
104 and SEQ ID NO: 372 to SEQ ID NO: 415). In a more preferred
embodiment, the peptidic linker that can link said catalytic domain
to the core TALE scaffold according to the method of the present
invention can be selected from the group consisting of NFS1 (SEQ ID
NO: 98), NFS2 (SEQ ID NO: 99) and CFS1 (SEQ ID NO: 100). In the
scope of the present invention is also encompassed the case where a
peptidic linker is not needed to fuse a catalytical domain to the
TALE scaffold in order to obtain a cTALEN according to the present
invention.
[0157] Depending from its structural composition [type of core TALE
scaffold, type of catalytic domain(s) with associated enzymatic
activities and eventually type of linker(s)], a compact TALEN
according to the present invention can comprise different levels of
separate enzymatic activities able to differently process DNA,
resulting in a global DNA processing efficiency for said compact
TALEN, each one of said different enzymatic activities having their
own DNA processing efficiency.
[0158] In another preferred embodiment, the compact TALEN according
to the present invention further comprises: [0159] (i) at least one
enhancer domain; [0160] (ii) Optionally one peptide linker to fuse
said enhancer domain to one part of said compact TALEN active
entity; thereby obtaining a compact TALEN entity with enhanced DNA
processing efficiency nearby a single double-stranded DNA target
sequence of interest, i.e. an enhanced compact TALEN.
[0161] In other words, said unique compact TALEN monomer further
comprises: [0162] (i) At least one enhancer domain; [0163] (ii)
Optionally one peptide linker to fuse said enhancer domain to one
part of said unique compact TALEN monomer active entity.
[0164] In another more preferred embodiment, said enhancer domain
is fused to N-terminus of the core TALE scaffold part of said
compact TALEN entity. In another more preferred embodiment, said
enhancer domain is fused to C-terminus of the core TALE scaffold
part of said compact TALEN entity. In another more preferred
embodiment, said enhancer domain is fused to the catalytic domain
part of said compact TALEN entity. In another more preferred
embodiment, said enhancer domain is fused between the N-terminus
part of the core TALE scaffold and the catalytic part of said
compact TALEN entity. In another more preferred embodiment, said
enhancer domain is fused between the C-terminus part of the core
TALE scaffold and the catalytic part of said compact TALEN entity.
In the scope of the present invention, it can be envisioned to
insert said catalytic domain and/or enhancer domain between two
parts of the engineered core TALE scaffold according to the
invention, each part comprising one set of RVDs. In this last case,
the number of RVDs for each engineered core TALE scaffold can be
the same or not. In other words, it can be envisioned to split said
core TALE scaffold of the present invention to insert one catalytic
domain and/or one enhancer domain between the resulting two parts
of said engineered core TALE scaffold.
[0165] In another preferred embodiment, said enhancer domain is
catalytically active or not, providing functional and/or structural
support to said compact TALEN entity. In a more preferred
embodiment, said enhancer domain consists of a protein domain
selected from the group consisting of MmeI, Colicin-E7
(CEA7_ECOLX), Colicin-E9, APFL, EndA, Endo I (END1_ECOLI), Human
Endo G (NUCG_HUMAN), Bovine Endo G (NUCG_BOVIN), R.HinP1I, I-BasI,
I-BmoI, I-HmuI, I-TevI, I-TevII, I-TevIII, I-TwoI, R.MspI, R.MvaI,
NucA, NucM, Vvn, Vvn_CLS, Staphylococcal nuclease (NUC_STAAU),
Staphylococcal nuclease (NUC_STAHY), Micrococcal nuclease
(NUC_SHIFL), Endonuclease yncB, Endodeoxyribonuclease I
(ENRN_BPT7), Metnase, Nb.BsrDI, BsrDI A, Nt.BspD6I (R.BspD6I large
subunit), ss.BspD6I (R.BspD6I small subunit), R.PleI, MIyI, AlwI,
Mva1269I, BsrI, BsmI, Nb.BtsCI, Nt.BtsCI, R1.BtsI, R2.BtsI, BbvCI
subunit 1, BbvCI subunit 2, Bpu10I alpha subunit, Bpu10I beta
subunit, BmrI, BfiI, I-CreI, hExol (EXO1_HUMAN), Yeast Exol
(EXO1_YEAST), E. coli Exol, Human TREX2, Mouse TREX1, Human TREX1,
Bovine TREX1, Rat TREX1, Human DNA2, Yeast DNA2 (DNA2_YEAST) and
VP16, as listed in Table 2 (SEQ ID NO: 10 to SEQ ID NO: 66 and SEQ
ID NO: 1, 366 & 367), a functional mutant, a variant or a
derivative of these protein domains thereof. In another more
preferred embodiment, said enhancer domain consists of a
catalytically active derivative of the protein domains listed above
and in Table 2, providing functional and/or structural support to
said compact TALEN entity. In another preferred embodiment, said
enhancer domain consists of a catalytically inactive derivative of
the protein domains listed above and in Table 2, providing
structural support to said compact TALEN entity. In another
preferred embodiment, said enhancer domain is selected from the
group consisting of I-TevI (SEQ ID NO: 20), ColE7 (SEQ ID NO: 11)
and NucA (SEQ ID NO: 26).
[0166] In a more preferred embodiment, said enhanced compact TALEN
according to the present invention can comprise a second enhancer
domain. In this embodiment, said second enhancer domain can have
the same characteristics than the first enhancer domain. In a more
preferred embodiment, said second enhancer domain provides
structural support to enhanced compact TALEN entity. In another
more preferred embodiment, said second enhancer domain provides
functional support to enhanced compact TALEN entity. In a more
preferred embodiment, said second enhancer domain provides
structural and functional supports to enhanced compact TALEN
entity. In a more preferred embodiment, said enhanced compact TALEN
entity comprises one catalytic domain and one enhancer domain. In
another more preferred embodiment said enhanced compact TALEN
entity comprises one catalytic domain and two enhancer domains. In
another more preferred embodiment said enhanced compact TALEN
entity comprises two catalytic domains and one enhancer domains. In
another more preferred embodiment said enhanced compact TALEN
entity comprises two catalytic domains and two enhancer
domains.
[0167] In a more preferred embodiment, said second enhancer domain
consists of a protein domain derived from a protein selected from
the group consisting of MmeI, Colicin-E7 (CEA7_ECOLX), Colicin-E9,
APFL, EndA, Endo I (END1_ECOLI), Human Endo G (NUCG_HUMAN), Bovine
Endo G (NUCG_BOVIN), R.HinP1I, I-BasI, I-BmoI, I-HmuI, I-TevI,
I-TevII, I-TevIII, I-TwoI, R.MspI, R.MvaI, NucA, NucM, Vvn,
Vvn_CLS, Staphylococcal nuclease (NUC_STAAU), Staphylococcal
nuclease (NUC_STAHY), Micrococcal nuclease (NUC_SHIFL),
Endonuclease yncB, Endodeoxyribonuclease I (ENRN_BPT7), Metnase,
Nb.BsrDI, BsrDI A, Nt.BspD6I (R.BspD6I large subunit), ss.BspD6I
(R.BspD6I small subunit), R.PleI, MlyI, AlwI, Mva1269I, BsrI, BsmI,
Nb.BtsCI, Nt.BtsCI, R1.BtsI, R2.BtsI, BbvCI subunit 1, BbvCI
subunit 2, Bpu10I alpha subunit, Bpu10I beta subunit, BmrI, BfiI,
I-CreI, hExol (EXO1_HUMAN), Yeast Exol (EXO1_YEAST), E. coli Exol,
Human TREX2, Mouse TREX1, Human TREX1, Bovine TREX1, Rat TREX1,
Human DNA2, Yeast DNA2 (DNA2_YEAST) and VP16, as listed in Table 2
(SEQ ID NO: 10 to SEQ ID NO: 66 and SEQ ID NO: 1, 366 & 367), a
functional mutant, a variant or a derivative of these protein
domains thereof. In another more preferred embodiment, said second
enhancer domain consists of a catalytically active derivative of
the protein domains listed above and in Table 2, providing
functional and/or structural support to said enhanced compact TALEN
entity. In another preferred embodiment, said second enhancer
domain consists of a catalytically inactive derivative of the
protein domains listed above and in Table 2, providing structural
support to said enhanced compact TALEN entity.
[0168] In another more preferred embodiment, any combinations of
catalytic and/or enhancer domains listed above, as non-limiting
examples, can be envisioned to be fused to said core TALE scaffold
providing structural and/or functional support to said compact
TALEN entity. More preferably, combinations of catalytic domains
listed in Table 14. Again more preferably, combinations of
catalytic domains selected from the group of TevI (SEQ ID NO: 20),
ColE7 (SEQ ID NO: 11) and NucA (SEQ ID NO: 26) can be envisioned.
Optionally, FokI (SEQ ID NO: 368) can be used in combination with
another catalytic domain according to the list of Tablet. Such
combinations of catalytic and/or enhancer domains can be envisioned
regarding the envisioned applications for using the method of the
present invention. Depending from its structural composition [type
of core TALE scaffold, type of catalytic domain(s) with associated
enzymatic activities, type of linker(s) and type of enhancer(s)
domains], an enhanced compact TALEN according to the present
invention can present different levels of separate enzymatic
activities able to differently process DNA, resulting in a global
DNA processing efficiency for said enhanced compact TALEN, each one
of said different enzymatic activities having their own DNA
processing efficiency.
[0169] In this preferred embodiment, the DNA processing efficiency
of the compact TALEN entity according to the present invention can
be enhanced by the engineering of at least one enhancer domain and
one peptidic linker thereby obtaining a compact TALEN entity with
enhanced DNA processing activity nearby a single double-stranded
DNA target sequence of interest, i.e. a enhanced compact TALEN
according to the present invention.
[0170] Depending from its structural composition, the global DNA
processing efficiency that is enhanced in said enhanced compact
TALEN according to the present invention, can have a dominant
enzymatic activity selected from the group consisting of a nuclease
activity, a polymerase activity, a kinase activity, a phosphatase
activity, a methylase activity, a topoisomerase activity, an
integrase activity, a transposase activity or a ligase activity as
non-limiting examples. In a more preferred embodiment, the global
DNA processing efficiency that is enhanced in said enhanced compact
TALEN according to the present invention is a combination of
different enzymatic activities selected from the group consisting
of a nuclease activity, a polymerase activity, a kinase activity, a
phosphatase activity, a methylase activity, a topoisomerase
activity, an integrase activity, a transposase activity or a ligase
activity as non-limiting examples. In a more preferred embodiment,
the global DNA processing efficiency that is enhanced in said
enhanced compact TALEN according to the present invention is one of
its different enzymatic activities selected from the group
consisting of a nuclease activity, a polymerase activity, a kinase
activity, a phosphatase activity, a methylase activity, a
topoisomerase activity, an integrase activity, a transposase
activity or a ligase activity as non-limiting examples. In this
case, the global DNA processing efficiency is equivalent to one DNA
processing activity amongst the enzymatic activities mentioned
above. In another more preferred embodiment, said DNA processing
activity of the compact TALEN entity which is enhanced by the
enhancer is a cleavase activity or a nickase activity or a
combination of both a cleavase activity and a nickase activity.
[0171] Enhancement of DNA processing efficiency of a compact TALEN
entity according to the present invention can be a consequence of a
structural support by said at least one enhancer domain. In a
preferred embodiment, said structural support enhances the binding
of a compact TALEN entity according to the invention for said DNA
target sequence compared to the binding of a starting compact TALEN
entity for the same DNA target sequence, thereby indirectly
assisting the catalytic domain(s) to obtain a compact TALEN entity
with enhanced DNA processing activity. In another preferred
embodiment, said structural support enhances the existing
catalytical activity of a compact TALEN entity for a DNA target
sequence compared to the binding of a starting compact TALEN entity
for the same DNA target sequence to obtain a compact TALEN entity
with enhanced DNA processing activity.
[0172] In another preferred embodiment, said enhancer according to
the present invention both enhances the binding of the compact
TALEN entity for said DNA target sequence and the catalytic
activity of the catalytic domain(s) to obtain a compact TALEN
entity with enhanced DNA processing activity. All these
non-limiting examples lead to a compact TALEN entity with enhanced
DNA processing efficiency for a DNA target sequence at a genomic
locus of interest, i.e. an enhanced compact TALEN according to the
present invention.
[0173] Enhancement of DNA processing efficiency of a compact TALEN
entity according to the present invention, compared to a starting
compact TALEN entity, can also be a consequence of a fuctional
support by said at least one enhancer domain. In a preferred
embodiment, said functional support can be the consequence of the
hydrolysis of additional phosphodiester bonds. In a more preferred
embodiment, said functional support can be the hydrolysis of
additional phosphodiester bonds by a protein domain derived from a
nuclease. In a more preferred embodiment, said functional support
can be the hydrolysis of additional phosphodiester bonds by a
protein domain derived from an endonuclease. In a more preferred
embodiment, said functional support can be the hydrolysis of
additional phosphodiester bonds by a protein domain derived from a
cleavase. In another more preferred embodiment, said functional
support can be the hydrolysis of additional phosphodiester bonds by
a protein domain derived from a nickase. In a more preferred
embodiment, said functional support can be the hydrolysis of
additional phosphodiester bonds by a protein domain derived from an
exonuclease.
[0174] In genome engineering experiments, the efficiency of
rare-cutting endonuclease, e.g. their ability to induce a desired
event (Homologous gene targeting, targeted mutagenesis, sequence
removal or excision) at a locus, depends on several parameters,
including the specific activity of the nuclease, probably the
accessibility of the target, and the efficacy and outcome of the
repair pathway(s) resulting in the desired event (homologous repair
for gene targeting, NHEJ pathways for targeted mutagenesis).
[0175] Cleavage by peptidic rare cutting endonucleases usually
generates cohesive ends, with 3' overhangs for LAGLIDADG
meganucleases (Chevalier and Stoddard 2001) and 5' overhangs for
Zinc Finger Nucleases (Smith, Bibikova et al. 2000). These ends,
which result from hydrolysis of phosphodiester bonds, can be
re-ligated in vivo by NHEJ in a seamless way (i.e. a scarless
re-ligation). The restoration of a cleavable target sequence allows
for a new cleavage event by the same endonuclease, and thus, a
series of futile cycles of cleavage and re-ligation events can take
place. Indirect evidences have shown that even in the yeast
Saccharomyces cerevisiae, such cycles could take place upon
continuous cleavage by the HO endonuclease (Lee, Paques et al.
1999). In mammalian cells, several experiment have shown that
perfect re-ligation of compatible cohesive ends resulting from two
independent but close I-SceI-induced DSBs is an efficient process
(Guirouilh-Barbat, Huck et al. 2004; Guirouilh-Barbat, Rass et al.
2007; Bennardo, Cheng et al. 2008; Bennardo, Gunn et al. 2009).
Absence of the Ku DNA repair protein does not significantly affect
the overall frequency of NHEJ events rejoining the ends from the
two DSBs; however it very strongly enhances the contribution of
imprecise NHEJ to the repair process in CHO immortalized cells and
mouse ES cells (Guirouilh-Barbat, Huck et al. 2004;
Guirouilh-Barbat, Rass et al. 2007; Bennardo, Cheng et al. 2008).
Furthermore, the absence of Ku stimulates I-SceI-induced events
such as imprecise NHEJ (Bennardo, Cheng et al. 2008), single-strand
annealing (Bennardo, Cheng et al. 2008) and gene conversion
(Pierce, Hu et al. 2001; Bennardo, Cheng et al. 2008) in mouse ES
cells. Similar observations shave been made with cells deficient
for the XRCC4 repair protein (Pierce, Hu et al. 2001;
Guirouilh-Barbat, Rass et al. 2007; Bennardo, Gunn et al. 2009)
(although XRCC4 deficiency affects the overal level of NHEJ in CHO
cells (Guirouilh-Barbat, Rass et al. 2007)) or for DNA-PK (Pierce,
Hu et al. 2001). In contrast, knock-down of CtIP has been shown to
suppresses "alt-NHEJ" (a Ku- and XRCC4-independent form of NHEJ
more prone to result in imprecise NHEJ), single-strand annealing
and gene conversion, while not affecting the overall level of
rejoining of two compatible ends generated by I-SceI (Bennardo,
Cheng et al. 2008). Thus, competition between different DSB repair
pathways can affect the spectrum or repair events resulting from a
nuclease-induced DSB.
[0176] In addition, DSB resection is important for certain DSB
pathways. Extensive DSB resection, resulting in the generation of
large single stranded regions (a few hundred nucleotides at least),
has been shown in yeast to initiate single strand annealing
(Sugawara and Haber 1992) and strand invasion, the ATP-dependant
step that initiates many homologous recombination events of DNA
duplex invasion by an homologous strand that (White and Haber 1990;
Sun, Treco et al. 1991) (for a review of mechanisms, see (Paques
and Haber 1999)). In eukaryotic cells DSB resection depends on
several proteins including BLM/Sgs1 and DNA2, EXOI, and the MRN
complex (Mre11, Rad50, Nbs1/Xrs2) and is thought to result from
different pathways. MRN is involved in a small scale resection
process, while two redundant pathways depending on BLM and DNA2 on
one hand, and on EXOI on another hand, would be involved in
extensive resection (Mimitou and Symington 2008; Nimonkar, Genschel
et al. 2011). In addition, processing ends involving a damaged
nucleotide (resulting from chemical cleavage or from a bulk
adduct), requires the CtIP/Sae2 protein together with RMN (Sartori,
Lukas et al. 2007; Buis, Wu et al. 2008; Hartsuiker, Mizuno et al.
2009). Over-expression of the Trex2 exonuclease was shown to
strongly stimulate imperfect NHEJ associated with loss of only a
few base pairs (Bennardo, Gunn et al. 2009), while it inhibited
various kinds of DNA repair events between distant sequences (such
as Single-strand annealing, NHEJ between ends from different
breaks, or NHEJ repair of a single DSB involving remote
micro-homologies). In the same study, it was suggested that Trex2
did resect the 3' overhangs let by I-SceI in a non processive way.
Thus, the type of stimulated pathway could in turn depend on the
type of resection (length of resection, single strand vs. double
strand, resection of 5' strand vs. 3' strand).
[0177] Thus, the efficiency of a compact TALEN, e.g. it ability to
produce a desired event such as targeted mutagenesis or homologous
gene targeting (see definition for full definition of "efficiency
of compact TALEN"), can be enhanced by an enhancement or
modification of its global DNA processing efficiency (see
definition for full definition of "global DNA processing
efficiency"), e.g. the global resultant or the overall result of
different separate enzymatic activities that said compact
TALEN.
[0178] In a preferred embodiment, enhancement of global DNA
processing efficiency of a compact TALEN entity according to the
present invention, compared to a starting compact TALEN entity, can
be the hydrolysis of additional phosphodiester bonds at the
cleavage site.
[0179] Said hydrolysis of additional phosphodiester bonds at the
cleavage site by said at least one enhancer according to the
invention can lead to different types of DSB resection affecting at
said DSB cleavage site, one single DNA strand or both DNA strands,
affecting either 5' overhangs ends, either 3' overhangs ends, or
both ends and depending on the length of said resection. Thus,
adding new nickase or cleavase activities to the existing cleavase
activity of a compact TALEN entity can enhance the efficiency of
the resulting enhanced compact TALEN according to the invention, at
a genomic locus of interest (FIG. 8B-8E). As a non-limiting
example, addition of two nickase activities on opposite strands
(FIG. 8D) or of a new cleavase activity generating a second DSB
(FIG. 8E) can result in a double-strand gap. As a consequence,
perfect religation is not possible anymore, and one or several
alternative repair outcomes such as imprecise NHEJ, Homologous
Recombination or SSA for instance, can be stimulated. As another
non-limiting example, the addition of a single nickase activity can
result in a single strand gap, and suppress the cohesivity of the
ends, which can also enhances the efficiency of the resulting
enhanced compact TALEN at a genomic locus of interest, according to
the invention, via stimulation of one or several alternative repair
outcomes mentioned above.
[0180] In this aspect of the present invention, enhancement of DNA
processing efficiency of a compact TALEN refers to the increase in
the detected level of said DNA processing efficiency, against a
target DNA sequence, of a compact TALEN in comparison to the
activity of a first compact TALEN against the same target DNA
sequence. Said first compact TALEN can be a starting compact TALEN,
or a compact TALEN that has already been engineered or an enhanced
compact TALEN according to the present invention. Several rounds of
enhancement can be envisioned from a starting compact TALEN or from
a starting enhanced compact TALEN.
[0181] In this aspect of the present invention, enhancement of the
DNA processing efficiency of the compact TALEN entity (or enhanced
compact TALEN) refers to the increase in the detected level of said
DNA processing efficiency against a target DNA sequence of interest
or nearby said DNA sequence of interest in comparison to the
efficiency of a first compact TALEN or starting compact TALEN
against or nearby the same target DNA sequence. In this case, the
starting compact TALEN is taken as the reference scaffold to
measure the DNA processing efficiency. Said enhanced compact TALEN
is an engineered compact TALEN comprising an enhancer domain
according to this aspect of the invention. Said enhanced compact
TALEN can also be taken as a reference scaffold for further
enhancement in said DNA processing efficiency. As a non-limiting
example, said DNA processing efficiency can result from a
cleavage-induced recombination generated by said enhanced compact
TALEN. In this case, said level of cleavage-induced recombination
can be determined, for instance, by a cell-based recombination
assay as described in the International PCT Application WO
2004/067736. Importantly, enhancement of efficacy in cells
(enhanced generation of targeted mutagenesis or targeted
recombination) can be, but is not necessarily associated with an
enhancement of the cleavage activity that could be detected in
certain in vitro assays. For example, additional phosphodiesterase
activities as described in FIG. 8 could barely affect the cleavage
profile, as detected by in vitro cleavage and separation of the
cleavage products on an electrophoresis gel. However, as explained
above, and in the legend of FIG. 8, the DSB ends generated in this
way could be more prone to induce detectable genomic rearrangements
such as targeted mutagenesis (by imprecise NHEJ) or homologous
recombination. Said enhancement in cleavage-induced recombination
of said enhanced compact TALEN is at least a 5% enhancement
compared to the starting scaffold or starting compact TALEN, more
preferably at least a 10% enhancement, again more preferably at
least a 15% enhancement, again more preferably at least a 20%
enhancement, again more preferably at least a 25% enhancement,
again more preferably a 50% enhancement, again more preferably a
enhancement greater than 50%, resulting in an enhancement of DNA
processing efficiency of said enhanced compact TALEN of at least 5%
compared to the starting scaffold or starting compact TALEN, more
preferably at least a 10% enhancement, again more preferably at
least a 15% enhancement, again more preferably at least a 20%
enhancement, again more preferably at least a 25% enhancement,
again more preferably a 50% enhancement, again more preferably a
enhancement greater than 50%.
[0182] In another preferred embodiment according to the method of
the present invention, the peptidic linker that can link said
enhancer domain to one part of said compact TALEN entity according
to the method of the present invention can be selected from the
group consisting of NFS1, NFS2, CFS1, RM2, BQY, QGPSG, LGPDGRKA,
1a8h.sub.--1, 1dnpA.sub.--1, 1d8cA.sub.--2, 1ckqA.sub.--3,
1sbp.sub.--1, 1ev7A.sub.--1, 1alo.sub.--3, 1amf.sub.--1,
1adjA.sub.--3, 1fcdC.sub.--1, 1a13.sub.--2, 1g3p.sub.--1,
1acc.sub.--3, 1ahjB.sub.--1, 1acc.sub.--1, 1af7.sub.--1,
1heiA.sub.--1, 1bia.sub.--2, 1igtB.sub.--1, 1nfkA.sub.--1,
1au7A.sub.--1, 1 bpoB.sub.--1, 1b0pA.sub.--2, 1c05A.sub.--2,
1gcb.sub.--1, 1bt3A.sub.--1, 1b3oB.sub.--2, 16vpA.sub.--6,
1dhx.sub.--1, 1b8aA.sub.--1 and 1qu6A.sub.--1 as listed in table 3
(SEQ ID NO: 67 to SEQ ID NO: 104 and SEQ ID NO: 372 to SEQ ID NO:
415). In a more preferred embodiment, the peptidic linker that can
said enhancer domain to one part of said compact TALEN entity
according to the method of the present invention can be selected
from the group consisting of NFS1 (SEQ ID NO: 98), NFS2 (SEQ ID NO:
99) and CFS1 (SEQ ID NO: 100). In the scope of the present
invention is also encompassed the case where a peptidic linker is
not needed to fuse one enhancer domain to one part of said compact
TALEN entity in order to obtain a enhanced compact TALEN according
to the present invention.
[0183] Depending from its structural composition [type of core TALE
scaffold, type of catalytic domain(s) with associated enzymatic
activities, type of enhancers and eventually type of linker(s)], a
compact TALEN or an enhanced compact TALEN according to the present
invention can comprise different levels of separate enzymatic
activities able to differently process DNA as mentioned above. By
adding new enzymatic activities to said compact TALEN or enhanced
compact TALEN or enhancing the DNA processing efficiency of one or
several of its constitutive enzymatic activities, one can enhance
the global DNA processing efficiency of one compact TALEN or
enhanced compact TALEN in comparison to a starting compact TALEN or
enhanced compact TALEN.
[0184] According to the present invention, compact TALENs are
designed to alleviate the need for multiple independent protein
moieties when targeting a DNA processing event. Importantly, the
requisite "spacer" region and dual target sites essential for the
function of current TALENs are unnecessary, as compact TALENs
according to the invention comprises a core TALE scaffold
containing only one DNA binding domain to target a specific single
double-stranded DNA target sequence of interest and process DNA
nearby said single double-stranded DNA target sequence of interest.
As each end of the core TALE scaffold is amenable to fusion, the
order (N- v.s C-terminal) of addition of the catalytic and
enhancement domains can vary with the application. In addition,
since the catalytic domain does not require specific DNA contacts,
there are no restrictions on regions surrounding the core TALE
scaffold, as non-limiting examples depicted in FIG. 5: (A)
N-terminal fusion construct to promote Homologous recombination
induced by a cleavase domain or by a nickase domain. (B) C-terminal
fusion construct with properties as in (A). (C) The attachment of
two catalytic domains to both ends of the core TALE scaffold allows
for dual cleavage with enhancement in NHEJ. Fusion junctions (N-vs.
C-terminal) and linker designs can vary with the application.
[0185] According to the present invention, compact TALENs can be
enhanced through the addition of a domain to promote existing or
alternate activities as non-limiting examples depicted in FIG. 6:
(A) A standard compact TALEN with an enhancer domain fused to the
C-terminus of its core TALE scaffold part. (B) The enhancer domain
is fused to the compact TALEN via the N-terminus of its catalytic
domain part. Such a configuration can be used to assist and/or
anchor the catalytic domain part near the DNA to increase DNA
processing activity. (C) The enhancer domain is sandwiched between
the catalytic domain part and the core TALE scaffold part. The
enhancer domain can promote communication between the flanking
domains (i.e. to assist in catalysis and/or DNA binding) or can be
used to overcome the requisite T nucleotide at position -1 of all
TALE-based targets. (D) The enhancer domain is used to functionally
replace the engineered core TALE scaffold N-terminal region. (E)
The enhancer domain is used to functionally replace the engineered
core TALE scaffold C-terminal region. Fusion junctions (N-vs.
C-terminal) and linker designs can vary with the application.
[0186] According to the present invention, the nature of the
catalytic domain(s) comprised in the compact TALEN and the enhanced
compact TALEN is application dependent. As a non-limiting example,
a nickase domain should allow for a higher HR/NHEJ ratio than a
cleavase domain, thereby being more agreeable for therapeutic
applications (McConnell Smith, Takeuchi et al. 2009; Metzger,
McConnell-Smith et al. 2011). For example, the coupling of a
cleavase domain on one side with a nickase domain on the other
could result in excision of a single-strand of DNA spanning the
binding region of a compact TALEN. The targeted generation of
extended single-strand overhangs could be applied in applications
that target DNA repair mechanisms. For targeted gene inactivation,
the use of two cleavase domains is then preferred. In another
preferred embodiment, the use of two nickase domains can be
favored. Furthermore, the invention relates to a method for
generating several distinct types of compact TALENs that can be
applied to applications ranging from targeted DNA cleavage to
targeted gene regulation.
[0187] The present invention also relates to methods for use of
said compact TALENs according to the invention for various
applications ranging from targeted DNA cleavage to targeted gene
regulation. In a preferred embodiment, the present invention
relates to a method for increasing targeted HR (and NHEJ) when
Double-Strand break activity is promoted in a compact TALEN
targeting a DNA target sequence according to the invention. In
another more preferred embodiment, the addition of at least two
catalytically active cleavase enhancer domains according to the
invention allows to increase Double-strand break-induced
mutagenesis by leading to a loss of genetic information and
preventing any scarless re-ligation of targeted genomic locus of
interest by NHEJ.
[0188] In another preferred embodiment, the present invention
relates to a method for increasing targeted HR with less NHEJ (i.e.
in a more conservative fashion) when Single-Strand Break activity
is promoted in a compact TALEN targeting a DNA target sequence
according to the invention.
[0189] In another preferred embodiment, the present invention
relates to a method for increasing excision of a single-strand of
DNA spanning the binding region of a compact TALEN when both one
cleavase enhancer domain and one nickase enhancer domain,
respectively, are fused to both N-terminus and C-terminus of a core
TALE scaffold according to the invention.
[0190] In another preferred embodiment, the present invention
relates to a method for treatment of a genetic disease caused by a
mutation in a specific single double-stranded DNA target sequence
in a gene, comprising administering to a subject in need thereof an
effective amount of a variant of a compact TALEN according to the
present invention.
[0191] In another preferred embodiment, the present invention
relates to a method for inserting a transgene into a specific
single double-stranded DNA target sequence of a genomic locus of a
cell, tissue or non-human animal, or a plant wherein at least one
compact TALEN of the present invention is transitory or not
introduced into said cell, tissue, non-human animal or plant.
[0192] In another embodiment, the present invention relates to a
method to modulate the activity of a compact TALEN when expressed
in a cell wherein said method comprises the step of introducing in
said cell an auxiliary domain modulating the activity of said
compact TALEN. In a preferred embodiment, the present invention
relates to a method which allows to have a temporal control of
activity of a compact TALEN when expressed in a cell by introducing
in said cell an auxiliary domain modulating the activity of said
compact TALEN once said compact TALEN achieved its activity (DNA
cleavage, DNA nicking or other DNA processing activities). In a
preferred embodiment, the present invention relates to a method to
inhibit the activity of a compact TALEN when expressed in a cell
wherein said method comprises the step of introducing in said cell
an auxiliary domain inhibiting the activity of said compact TALEN.
In a more preferred embodiment, the catalytic domain of said
compact TALEN is NucA (SEQ ID NO: 26) and said auxiliary domain is
NuiA (SEQ ID NO: 229), a functional mutant, a variant or a
derivative thereof. In another more preferred embodiment, the
catalytic domain of said compact TALEN is ColE7 (SEQ ID NO: 11) and
said auxiliary domain is Im7 (SEQ ID NO: 230), a functional mutant,
a variant or a derivative thereof.
[0193] Is also encompassed in the scope of the present invention a
recombinant polynucleotide encoding a compact TALEN, a dual compact
TALEN, or an enhanced compact TALEN according to the present
invention. Is also encompassed in the scope of the present
invention, a vector comprising a recombinant polynucleotide
encoding for a compact TALEN or an enhanced compact TALEN according
to the present invention.
[0194] Is also encompassed in the scope of the present invention, a
host cell which comprises a vector and/or a recombinant
polynucleotide encoding for a compact TALEN or an enhanced compact
TALEN according to the present invention.
[0195] Is also encompassed in the scope of the present invention, a
non-human transgenic animal comprising a vector and/or a
recombinant polynucleotide encoding for a compact TALEN or an
enhanced compact TALEN according to the present invention.
[0196] Is also encompassed in the scope of the present invention, a
transgenic plant comprising a vector and/or a recombinant
polynucleotide encoding for a compact TALEN or an enhanced compact
TALEN according to the present invention.
[0197] The present invention also relates to kits used to implement
the method according to the present invention. More preferably, is
encompassed in the scope of the present invention, a kit comprising
a compact TALEN or an enhanced compact TALEN according to the
present invention and instructions for use said kit in enhancing
DNA processing efficiency of a single double-stranded DNA target
sequence of interest.
[0198] For purposes of therapy, the compact TALENs of the present
invention and a pharmaceutically acceptable excipient are
administered in a therapeutically effective amount. Such a
combination is said to be administered in a "therapeutically
effective amount" if the amount administered is physiologically
significant. An agent is physiologically significant if its
presence results in a detectable change in the physiology of the
recipient. In the present context, an agent is physiologically
significant if its presence results in a decrease in the severity
of one or more symptoms of the targeted disease and in a genome
correction of the lesion or abnormality. Vectors comprising
targeting DNA and/or nucleic acid encoding a compact TALEN can be
introduced into a cell by a variety of methods (e.g., injection,
direct uptake, projectile bombardment, liposomes, electroporation).
Compact TALENs can be stably or transiently expressed into cells
using expression vectors. Techniques of expression in eukaryotic
cells are well known to those in the art. (See Current Protocols in
Human Genetics: Chapter 12 "Vectors For Gene Therapy" & Chapter
13 "Delivery Systems for Gene Therapy").
[0199] In one further aspect of the present invention, the compact
TALEN of the present invention is substantially non-immunogenic,
i.e., engender little or no adverse immunological response. A
variety of methods for ameliorating or eliminating deleterious
immunological reactions of this sort can be used in accordance with
the invention. In a preferred embodiment, the compact TALEN is
substantially free of N-formyl methionine. Another way to avoid
unwanted immunological reactions is to conjugate compact TALEN to
polyethylene glycol ("PEG") or polypropylene glycol ("PPG")
(preferably of 500 to 20,000 daltons average molecular weight
(MW)). Conjugation with PEG or PPG, as described by Davis et al.
(U.S. Pat. No. 4,179,337) for example, can provide non-immunogenic,
physiologically active, water soluble compact TALEN conjugates with
anti-viral activity. Similar methods also using a
polyethylene--polypropylene glycol copolymer are described in
Saifer et al. (U.S. Pat. No. 5,006,333).
[0200] In another aspect of the present invention is a composition
comprising a compact TALEN or an enhanced compact TALEN according
to the present invention and a carrier. More preferably, is a
pharmaceutical composition comprising a compact TALEN or an
enhanced compact TALEN according to the present invention and a
pharmaceutically active carrier known in the state of the art.
[0201] In the scope of the present invention and for all the
applications mentioned above, it can be envisioned to use more than
one compact TALEN (i.e. one compact TALEN active entity) or more
than one enhanced compact TALENs (i.e. one enhanced compact TALEN
active entity) for DNA processing according to the invention. In a
preferred embodiment, two different compact TALENs or two enhanced
compact TALENs can be used. In this embodiment, as non-limiting
examples, said two different compact TALENs can comprise the same
core TALE scaffold or not; said two different compact TALENs can
comprise the same set of Repeat Variable Dipeptides or not; said
two different compact TALENs can comprise the same catalytic domain
or not. When two identical compact TALENs active entities are used
for DNA processing according to the invention, they can be
considered as a homodimeric pair of compact TALENs active entities.
When two non identical compact TALENs active entities are used for
DNA processing according to the invention, they can be considered
as a heterodimeric pair of compact TALENs active entities. As
non-limiting example, when two compact TALEN according to the
present invention are used, one of the compact TALEN can modulate
the activity of the other one, leading for instance to an enhanced
DNA processing event compared to the same DNA processing event
achieved by only one compact TALEN; in this non-limiting example, a
Trans-TALEN modulates and enhances the catalytic activity of an
initial compact TALEN.
[0202] In another preferred embodiment, three compact TALENs or
three enhanced compact TALENs can be used. In another preferred
embodiment, more than three compact TALENs or three enhanced
compact TALENs can be used for DNA processing according to the
invention. In another preferred embodiment, a combination of
compact TALENs and enhanced compact TALENs can be used for DNA
processing according to the invention. As a non-limiting example,
one compact TALEN and one enhanced compact TALEN can be used. As
another non-limiting example, one compact TALEN and one
dual-cleavage compact TALEN can be used. In another preferred
embodiment, a combination of compact TALENs, enhanced compact
TALENs and dual-cleavage compact TALENs can be used, said compact
TALENs comprising the same catalytic domain or not, the same core
TALE scaffold or not. When several compact TALENs have to be used,
DNA target sequence for each compact TALENs of the combination to
be used can be located on a same endogenous genomic DNA locus of
interest or not. Said DNA target sequences can be located at an
approximative distance of 1000 base pairs (bps). More preferably,
said DNA target sequences can be located at an approximative
distance of 500 bps or 200 bps, or 100 bps, or 50 bps, or bps, 19
bps, 18 bps, 17 bps, 16 bps, 15 bps, 14 bps, 13 bps, 12 bps, 11
bps, 10 bps, 9 bps, 8 bps, 7 bps, 6 bps, 5 bps, 4 bps, 3 bps, 2
bps, 1 bp. Said DNA target sequences located at distances mentioned
above are "nearby" DNA sequences in reference to the target DNA
sequence for DNA processing according to the present invention.
[0203] In another preferred embodiment, two compact TALENs active
entities can be used as a way of achieving two different DNA
processing activities nearby a DNA target sequence according to the
invention. As a non-limiting example, two compact TALENs targeting
said DNA sequence or DNA sequences nearby said targeted DNA
sequence and comprising each one a nickase-derived catalytic domain
can be used; in this case, this use of two compact TALENs active
entities can represent an alternative way of achieving a Double
Strand Break nearby a said DNA target sequence, compared to the use
of one compact TALEN targeting said DNA sequence and comprising a
cleavase-derived catalytic domain, or not. As another non-limiting
example, one compact TALEN comprising a cleavase-derived domain and
one compact TALEN comprising an exonuclease-derived domain can be
used to make a Double Strand Break and create a gap, respectively,
to achieve an imprecise NHEJ event at the genomic locus of interest
comprising said DNA target sequence. In this case, even if each
compact TALEN forming this heterodimeric pair of compact TALENs is
active by itself, each of these active entities is dependent of the
other one to achieve the wanted resulting DNA processing activity.
Indeed, in this particular case, the wanted resulting activity is a
gap created by the exonuclease activity, said exonuclease activity
being possible only from the Double Strand Break achieved by the
cleavase domain of the other compact TALENs. In the scope of the
present invention, is also envisioned the case where two identical
compact TALEN active entities (a homodimeric pair of compact
TALENs) are dependent each other to achieve a wanted resulting DNA
processing activity.
DEFINITIONS
[0204] Amino acid residues in a polypeptide sequence are designated
herein according to the one-letter code, in which, for example, Q
means Gln or Glutamine residue, R means Arg or Arginine residue and
D means Asp or Aspartic acid residue. [0205] Amino acid
substitution means the replacement of one amino acid residue with
another, for instance the replacement of an Arginine residue with a
Glutamine residue in a peptide sequence is an amino acid
substitution. [0206] Enhanced/increased/improved DNA processing
activity, refers to an increase in the detected level of a given
compact TALEN or enhanced compact TALEN associated DNA processing
activity against a target DNA sequence or DNA target sequence by a
second compact TALEN or enhanced compact TALEN in comparison to the
activity of a first compact TALEN or enhanced compact TALEN against
the target DNA sequence. The second compact TALEN or enhanced
compact TALEN can be a variant of the first one and can comprise
one or more substituted amino acid residues in comparison to the
first compact TALEN or enhanced compact TALEN. The second compact
TALEN or enhanced compact TALEN can be a variant of the first one
and can comprise one or more catalytic and/or enhancer domains in
comparison to said first compact TALEN or enhanced compact TALEN.
This definition more broadly applies for other endonucleases and
rare-cutting endonucleases. [0207] DNA processing activity refers
to a particular/given enzymatic activity of said compact TALEN or
enhanced compact TALEN or more broadly to qualify the enzymatic
activity of a rare-cutting endonuclease. Said DNA processing
activity can refer to a cleavage activity, either a cleavase
activity either a nickase activity, more broadly a nuclease
activity but also a polymerase activity, a kinase activity, a
phosphatase activity, a methylase activity, a topoisomerase
activity, an integrase activity, a transposase activity or a ligase
activity as non-limiting examples. In the scope of this definition,
said given DNA processing activity of a particular enzymatic
activity can also be described as DNA processing efficiency of said
particular enzymatic activity. Methods for enhancing compact TALEN
or enhanced compact TALEN DNA processing activity according to this
definition are encompassed in the present invention. [0208] Global
DNA processing efficiency describes, for a compact TALEN or an
enhanced compact TALEN according to the present invention, the
global resultant or the overall effect of different separate
enzymatic activities that said compact TALEN or enhanced compact
TALEN can comprise. According to these different separate enzymatic
activities, a compact TALEN or an enhanced compact TALEN presents a
global capacity to process DNA nearby a target sequence in a
genomic locus of interest, i.e. a global DNA processing efficiency.
Said global DNA processing efficiency can qualify or rank a second
given compact TALEN or enhanced compact TALEN in comparison to a
first given compact TALEN or enhanced compact TALEN. Depending on
said compact TALENs or enhanced compact TALENs structural
composition [type of core TALE scaffold, type of catalytic
domain(s) with associated enzymatic activities, eventually type of
linker(s) and type of enhancer(s) domains], said global DNA
processing efficiency can refer to only one enzymatic activity, two
enzymatic activities, three enzymatic activities, four enzymatic
activities or more than four enzymatic activities. Said global DNA
processing efficiency can refer to the sum of individual enzymatic
activities. Said global DNA processing efficiency can refer to the
synergy or combined effect of different enzymatic activities
comprised in a given compact TALEN or enhanced compact TALEN. An
enhancement of the DNA processing efficiency of a compact TALEN
according to the present invention can reflect a synergy, an
enhanced combined effect, resulting in an enhanced compact TALEN
characterized by a global DNA processing efficiency that is greater
than the sum of respective DNA processing efficiencies of separate
starting compact TALEN or than the sum of respective DNA processing
efficiencies of separate enzymatic activities comprised in a same
starting compact TALEN. [0209] Efficiency of a rare-cutting
endonuclease according to the present invention is the property for
said rare-cutting endonuclease of producing a desired event. This
desired event can be for example Homologous gene targeting,
targeted mutagenesis, or sequence removal or excision. The
efficiency of the desired event depends on several parameters,
including the specific activity of the nuclease and the repair
pathway(s) resulting in the desired event (efficacy of homologous
repair for gene targeting, efficacy and outcome of NHEJ pathways
for targeted mutagenesis). Efficiency of a rare cutting
endonuclease for a locus is intended to mean its ability to produce
a desired event at this locus. Efficiency of a rare cutting
endonuclease for a target is intended to mean its ability to
produce a desired event as a consequence of cleavage of this
target. [0210] Nucleotides are designated as follows: one-letter
code is used for designating the base of a nucleoside: a is
adenine, t is thymine, c is cytosine, and g is guanine. For the
degenerated nucleotides, r represents g or a (purine nucleotides),
k represents g or t, s represents g or c, w represents a or t, m
represents a or c, y represents t or c (pyrimidine nucleotides), d
represents g, a or t, v represents g, a or c, b represents g, t or
c, h represents a, t or c, and n represents g, a, t or c. [0211] by
"meganuclease", is intended a rare-cutting endonuclease subtype
having a double-stranded DNA target sequence greater than 12 bp.
Said meganuclease is either a dimeric enzyme, wherein each domain
is on a monomer or a monomeric enzyme comprising the two domains on
a single polypeptide. [0212] by "meganuclease domain" is intended
the region which interacts with one half of the DNA target of a
meganuclease and is able to associate with the other domain of the
same meganuclease which interacts with the other half of the DNA
target to form a functional meganuclease able to cleave said DNA
target. [0213] by "endonuclease variant", "rare-cutting
endonuclease variant", "chimeric rare-cutting endonuclease variant"
or "meganuclease variant" or "compact TALEN variant", or "enhanced
compact TALEN variant" or "dual cleavage compact TALEN variant" or
"variant" it is intended an endonuclease, rare-cutting
endonuclease, chimeric rare-cutting endonuclease, meganuclease, or
compact TALEN, enhanced compact TALEN, dual cleavage compact TALEN
obtained by replacement of at least one residue in the amino acid
sequence of the parent endonuclease, rare-cutting endonuclease,
chimeric rare-cutting endonuclease, meganuclease or compact TALEN,
enhanced compact TALEN, dual cleavage compact TALEN with at least a
different amino acid. "Variant" designation also applies for
instance for an enhanced compact TALEN comprising at least one
supplementary protein domain (catalytic or enhancer domain) in
comparison to the starting compact TALEN entity. Are also
encompassed in the scope of the present definition, variants and
protein domains comprised in these variants which present a
sequence with high percentage of identity or high percentage of
homology with sequences of compact TALENs, enhanced compact TALENs,
dual-cleavage compact TALENs or protein domains and polypeptides
according to the present invention, at nucleotidic or polypeptidic
levels. By high percentage of identity or high percentage of
homology it is intended 60%, more preferably 70%, more preferably
75%, more preferably 80%, more preferably 85%, more preferably 90%,
more preferably 95, more preferably 97%, more preferably 99% or any
integer comprised between 60% and 99%. [0214] by "peptide linker",
"peptidic linker" or "peptide spacer" it is intended to mean a
peptide sequence which allows the connection of different monomers
in a fusion protein and the adoption of the correct conformation
for said fusion protein activity and which does not alter the
specificity of either of the monomers for their targets. Peptide
linkers can be of various sizes, from 3 amino acids to 50 amino
acids as a non limiting indicative range. Peptide linkers can also
be structured or unstructured. [0215] by "related to", particularly
in the expression "one cell type related to the chosen cell type or
organism", is intended a cell type or an organism sharing
characteristics with said chosen cell type or said chosen organism;
this cell type or organism related to the chosen cell type or
organism, can be derived from said chosen cell type or organism or
not. [0216] by "subdomain" it is intended the region of a LAGLIDADG
homing endonuclease core domain which interacts with a distinct
part of a homing endonuclease DNA target half-site. [0217] by
"targeting DNA construct/minimal repair matrix/repair matrix" it is
intended to mean a DNA construct comprising a first and second
portion that are homologous to regions 5' and 3' of the DNA target
in situ. The DNA construct also comprises a third portion
positioned between the first and second portion which comprise some
homology with the corresponding DNA sequence in situ or
alternatively comprise no homology with the regions 5' and 3' of
the DNA target in situ. Following cleavage of the DNA target, a
homologous recombination event is stimulated between the genome
containing the targeted gene comprised in the locus of interest and
the repair matrix, wherein the genomic sequence containing the DNA
target is replaced by the third portion of the repair matrix and a
variable part of the first and second portions of the repair
matrix. [0218] by "functional variant" is intended a catalytically
active variant of a protein, such variant can have additional
properties compared to its parent protein. As a non-limiting
example, a functional variant of a meganuclease can be able to
cleave a DNA target sequence, preferably said target being a new
target which is not cleaved by the parent meganuclease. This
definition also applies to compact TALENs, enhanced compact TALENs,
dual-cleavage compact TALENs or protein domains that constitute
such TALENs according to the present invention. Are also
encompassed in the scope of the present definition, functional
variants, polypeptides and protein domains comprised in these
molecules which present a sequence with high percentage of identity
or high percentage of homology with sequences of compact TALENs,
enhanced compact TALENs, dual-cleavage compact TALENs or protein
domains and polypeptides according to the present invention, at
nucleotidic or polypeptidic levels. By high percentage of identity
or high percentage of homology it is intended 60%, more preferably
70%, more preferably 75%, more preferably 80%, more preferably 85%,
more preferably 90%, more preferably 95, more preferably 97%, more
preferably 99% or any integer comprised between 60% and 99%. [0219]
by "derived from" or "derivative(s)" it is intended to mean for
instance a meganuclease variant which is created from a parent
meganuclease and hence the peptide sequence of the meganuclease
variant is related to (primary sequence level) but derived from
(mutations) the peptide sequence of the parent meganuclease. In
this definition, mutations encompass deletions or insertions of
several amino acid residues; as non-limiting example, a truncated
variant of an I-CreI meganuclease is considered as a scaffold
derived from I-CreI meganuclease. This expression can also apply to
compact TALENs, enhanced compact TALENs, dual-cleavage compact
TALENs or protein domains that constitute such TALENs according to
the present invention. Are also encompassed in the scope of the
present definition, derivatives of compact TALENs, enhanced compact
TALENs, dual-cleavage compact TALENs or protein domains and
derivatives of polypeptides according to the present invention
which present a sequence with high percentage of identity or high
percentage of homology with sequences of compact TALENs, enhanced
compact TALENs, dual-cleavage compact TALENs or protein domains and
polypeptides according to the present invention, at nucleotidic or
polypeptidic levels. By high percentage of identity or high
percentage of homology it is intended 60%, more preferably 70%,
more preferably 75%, more preferably 80%, more preferably 85%, more
preferably 90%, more preferably 95, more preferably 97%, more
preferably 99% or any integer comprised between 60% and 99%. [0220]
by "I-CreI" is intended the wild-type I-CreI having the sequence of
pdb accession code 1g9y, corresponding to the sequence SEQ ID NO: 1
in the sequence listing. In the present patent application, I-CreI
variants described can comprise an additional Alanine after the
first Methionine of the wild type I-CreI sequence (SEQ ID NO: 1).
These variants may also comprise two additional Alanine residues
and an Aspartic Acid residue after the final Proline of the wild
type I-CreI sequence as shown in SEQ ID NO: 106. These additional
residues do not affect the properties of the enzyme and to avoid
confusion these additional residues do not affect the numeration of
the residues in I-CreI or a variant referred in the present patent
application, as these references exclusively refer to residues of
the wild type I-CreI enzyme (SEQ ID NO: 1) as present in the
variant, so for instance residue 2 of I-CreI is in fact residue 3
of a variant which comprises an additional Alanine after the first
Methionine. [0221] by compact TALEN, enhanced compact TALEN,
dual-cleavage compact TALEN with novel specificity is intended a
variant of these proteins having a pattern of cleaved targets
different from that of their respective parent compact TALENs,
enhanced compact TALENs, dual-cleavage compact TALENs. The terms
"novel specificity", "modified specificity", "novel cleavage
specificity", "novel substrate specificity" which are equivalent
and used indifferently, refer to the specificity of the variant
towards the nucleotides of the DNA target sequence. [0222] by
"I-CreI site" is intended a 22 to 24 by double-stranded DNA
sequence which is cleaved by I-Crel. I-CreI sites include the
wild-type non-palindromic I-CreI homing site and the derived
palindromic sequences such as the sequence
5'-t.sub.-12c.sub.-11a.sub.-10a.sub.-9a.sub.-8a.sub.-7c.sub.-6g.sub.-5t.s-
ub.-4c.sub.-3g.sub.-2t.sub.-1a.sub.+1c.sub.+2g.sub.+3a.sub.+4c.sub.+5g.sub-
.+6t.sub.+7t.sub.+8t.sub.+9t.sub.+10g.sub.+11a.sub.+12 (SEQ ID NO:
2), also called C1221 or C1221 target. [0223] by "domain" or "core
domain" is intended the "LAGLIDADG homing endonuclease core domain"
which is the characteristic 413413a fold of the homing
endonucleases of the LAGLIDADG family, corresponding to a sequence
of about one hundred amino acid residues. Said domain comprises
four beta-strands
(.beta..sub.1.beta..sub.2.beta..sub.3.beta..sub.4) folded in an
anti-parallel beta-sheet which interacts with one half of the DNA
target. This domain is able to associate with another LAGLIDADG
homing endonuclease core domain which interacts with the other half
of the DNA target to form a functional endonuclease able to cleave
said DNA target. For example, in the case of the dimeric homing
endonuclease I-CreI (163 amino acids), the LAGLIDADG homing
endonuclease core domain corresponds to the residues 6 to 94.
[0224] by "beta-hairpin" is intended two consecutive beta-strands
of the antiparallel beta-sheet of a LAGLIDADG homing endonuclease
core domain (.beta..sub.1.beta..sub.2 or .beta..sub.3.beta..sub.4)
which are connected by a loop or a turn. [0225] by "single-chain
meganuclease", "single-chain chimeric meganuclease", "single-chain
meganuclease derivative", "single-chain chimeric mega nuclease
derivative" or "single-chain derivative" is intended a meganuclease
comprising two LAGLIDADG homing endonuclease domains or core
domains linked by a peptidic spacer. The single-chain meganuclease
is able to cleave a chimeric DNA target sequence comprising one
different half of each parent meganuclease target sequence. [0226]
by "DNA target", "DNA target sequence", "target DNA sequence",
"target sequence", "target-site", "target", "site", "site of
interest", "recognition site", "polynucleotide recognition site",
"recognition sequence", "homing recognition site", "homing site",
"cleavage site" is intended a double-stranded palindromic,
partially palindromic (pseudo-palindromic) or non-palindromic
polynucleotide sequence that is recognized and can be cleaved by a
LAGLIDADG homing endonuclease such as I-Crel, or a variant, or a
single-chain chimeric meganuclease derived from I-Crel. Said DNA
target sequence can be qualified as "cleavable" by an endonuclease,
rare-cutting endonuclease, chimeric rare-cutting endonuclease or
meganuclease when recognized within a genomic sequence and known to
correspond to the DNA target sequence of a given endonuclease,
rare-cutting endonuclease, chimeric rare-cutting endonuclease or
meganuclease or a variant of such endonuclease, rare-cutting
endonuclease, chimeric rare-cutting endonuclease or meganuclease.
These terms refer to a specific DNA location, preferably a genomic
location, but also a portion of genetic material that can exist
independently to the main body of genetic material such as
plasmids, episomes, virus, transposons or in organelles such as
mitochondria or chloroplasts as non-limiting examples, at which a
double stranded break (cleavage) can be induced by the
endonuclease, rare-cutting endonuclease, chimeric rare-cutting
endonuclease or meganuclease. For the LAGLIDADG subfamily of
rare-cutting endonucleases, the DNA target is defined by the 5' to
3' sequence of one strand of the double-stranded polynucleotide, as
indicate above for C1221 (SEQ ID NO: 2). Cleavage of the DNA target
can occur at the nucleotides at positions +2 and -2, respectively
for the sense and the antisense strand. Unless otherwise indicated,
the position at which cleavage of the DNA target by an
I-CreI-derived variant can occur, corresponds to the cleavage site
on the sense strand of the DNA target. In the particular case of
compact TALENs, a subclass of chimeric rare-cutting endonucleases,
the following expressions "DNA target", "DNA target sequence",
"target DNA sequence", "target sequence", "target-site", "target",
"site", "site of interest", "recognition site", "polynucleotide
recognition site", and "recognition sequence" can apply to qualify
their specific DNA target sequence with the particularity that said
specific DNA target sequence recognized by the compact TALEN
according to the invention is the one or not that is processed
and/or cut by the compact TALEN. A compact TALEN, an enhanced
compact TALEN or a dual cleavage compact TALEN according to the
present invention can process and/or cut DNA within said specific
DNA target sequence. A compact TALEN, an enhanced compact TALEN or
a dual cleavage compact TALEN can also process and/or cut DNA
outside said specific DNA target sequence. [0227] By "DNA nearby
said specific DNA target sequence" or by "DNA nearby" is intended
DNA sequence or sequences located within or outside said specific
DNA target sequence. Are also intended DNA sequence or sequences
bound by a compact TALEN or an enhanced compact TALEN at said
specific DNA target sequence location or DNA located at a 5' or 3'
distance of 1-100 base pairs (bps), 1-50 base pairs (bps) or 1-25
base pairs (bps) from said specific DNA target sequence.
[0228] When several compact TALENs have to be used in a particular
genome engineering application, DNA target sequence for each
compact TALENs of the combination to be used can be located on a
same endogenous genomic DNA locus of interest or not. Said DNA
target sequences can be located at an approximative distance of
1-1000 base pairs (bps), more preferably 1-500 bps, more preferably
1-100 bps, more preferably 1-100 bps, more preferably 1-50 bps,
more preferably 1-25 bps, more preferably 1-10 bps. In another
embodiment, said DNA target sequence for each compact TALENs of the
combination to be used can be located on the same DNA strand or
not. Said DNA target sequences located at distances mentioned above
are "nearby" DNA sequences in reference to the target DNA sequence
for DNA processing according to the present invention. [0229] by
"single double-stranded DNA target sequence" is intended a
compact-TALEN or enhanced compact TALEN or dual-cleavage compact
TALEN binding site. The recognition DNA binding site of a
compact-TALEN or enhanced compact TALEN or dual-cleavage compact
TALEN can be ranging from 12 to 100 base pairs (bp) in length,
usually greater than 12 bps in length. [0230] by "DNA target
half-site", "half cleavage site" or half-site" is intended the
portion of the DNA target which is bound by each LAGLIDADG homing
endonuclease core domain. [0231] The term "endonuclease" refers to
any wild-type or variant enzyme capable of catalyzing the
hydrolysis (cleavage) of bonds between nucleic acids within a DNA
or RNA molecule, preferably a DNA molecule. Endonucleases can be
classified as rare-cutting endonucleases when having typically a
polynucleotide recognition greater than 12 base pairs (bp) in
length, more preferably of 14-45 bp. Rare-cutting endonucleases
significantly increase HR by inducing DNA double-strand breaks
(DSBs) at a defined locus (Rouet, Smih et al. 1994; Rouet, Smih et
al. 1994; Choulika, Perrin et al. 1995; Pingoud and Silva 2007).
Rare-cutting endonucleases can for example be a homing endonuclease
(Paques and Duchateau 2007), a chimeric Zinc-Finger nuclease (ZFN)
resulting from the fusion of engineered zinc-finger domains with
the catalytic domain of a restriction enzyme such as FokI (Porteus
and Carroll 2005) or a chemical endonuclease (Eisenschmidt, Lanio
et al. 2005; Arimondo, Thomas et al. 2006; Simon, Cannata et al.
2008). In chemical endonucleases, a chemical or peptidic cleaver is
conjugated either to a polymer of nucleic acids or to another DNA
recognizing a specific target sequence, thereby targeting the
cleavage activity to a specific sequence. Chemical endonucleases
also encompass synthetic nucleases like conjugates of
orthophenanthroline, a DNA cleaving molecule, and triplex-forming
oligonucleotides (TFOs), known to bind specific DNA sequences
(Kalish and Glazer 2005). Such chemical endonucleases are comprised
in the term "endonuclease" according to the present invention.
[0232] Rare-cutting endonucleases can also be for example TALENs, a
new class of chimeric nucleases using a FokI catalytic domain and a
DNA binding domain derived from Transcription Activator Like
Effector (TALE), a family of proteins used in the infection process
by plant pathogens of the Xanthomonas genus (Boch, Scholze et al.
2009; Boch, Scholze et al. 2009; Moscou and Bogdanove 2009; Moscou
and Bogdanove 2009; Christian, Cermak et al. 2010; Christian,
Cermak et al. 2010; Li, Huang et al. 2010; Li, Huang et al. 2011).
The functional layout of a FokI-based TALE-nuclease (TALEN) is
essentially that of a ZFN, with the Zinc-finger DNA binding domain
being replaced by the TALE domain. As such, DNA cleavage by a TALEN
requires two DNA recognition regions flanking an unspecific central
region. Rare-cutting endonucleases encompassed in the present
invention can also be derived from TALENs. The authors of the
present invention have developed a new type of TALENs that can be
engineered to specifically recognize and process target DNA
efficiently. These novel "compact TALENs" (cTALENs) do not require
dimerization for DNA processing activity, thereby alleviating the
need for "dual" target sites with intervening DNA "spacers"; these
compact TALENs can be seen as one subclass of rare-cutting
endonucleases or chimeric rare-cutting endonucleases according to
the present invention.
[0233] Rare-cutting endonuclease can be a homing endonuclease, also
known under the name of meganuclease. Such homing endonucleases are
well-known to the art (Stoddard 2005). Homing endonucleases
recognize a DNA target sequence and generate a single- or
double-strand break. Homing endonucleases are highly specific,
recognizing DNA target sites ranging from 12 to 45 base pairs (bp)
in length, usually ranging from 14 to 40 by in length. The homing
endonuclease according to the invention may for example correspond
to a LAGLIDADG endonuclease, to a HNH endonuclease, or to a GIY-YIG
endonuclease.
[0234] In the wild, meganucleases are essentially represented by
homing endonucleases. Homing Endonucleases (HEs) are a widespread
family of natural meganucleases including hundreds of proteins
families (Chevalier and Stoddard 2001). These proteins are encoded
by mobile genetic elements which propagate by a process called
"homing": the endonuclease cleaves a cognate allele from which the
mobile element is absent, thereby stimulating a homologous
recombination event that duplicates the mobile DNA into the
recipient locus. Given their exceptional cleavage properties in
terms of efficacy and specificity, they could represent ideal
scaffolds to derive novel, highly specific endonucleases.
[0235] HEs belong to four major families. The LAGLIDADG family,
named after a conserved peptidic motif involved in the catalytic
center, is the most widespread and the best characterized group.
Seven structures are now available. Whereas most proteins from this
family are monomeric and display two LAGLIDADG motifs, a few have
only one motif, and thus dimerize to cleave palindromic or
pseudo-palindromic target sequences.
[0236] Although the LAGLIDADG peptide is the only conserved region
among members of the family, these proteins share a very similar
architecture. The catalytic core is flanked by two DNA-binding
domains with a perfect two-fold symmetry for homodimers such as
I-CreI (Chevalier, Monnat et al. 2001), I-MsoI (Chevalier, Turmel
et al. 2003) and I-CeuI (Spiegel, Chevalier et al. 2006) and with a
pseudo symmetry for monomers such as I-SceI (Moure, Gimble et al.
2003), I-DmoI (Silva, Dalgaard et al. 1999) or I-AniI (Bolduc,
Spiegel et al. 2003). Both monomers and both domains (for monomeric
proteins) contribute to the catalytic core, organized around
divalent cations. Just above the catalytic core, the two LAGLIDADG
peptides also play an essential role in the dimerization interface.
DNA binding depends on two typical saddle-shaped
.alpha..beta..beta..alpha..beta..beta..alpha. folds, sitting on the
DNA major groove. Other domains can be found, for example in
inteins such as PI-PfuI (Ichiyanagi, Ishino et al. 2000) and
PI-SceI (Moure, Gimble et al. 2002), whose protein splicing domain
is also involved in DNA binding.
[0237] The making of functional chimeric meganucleases, by fusing
the N-terminal I-DmoI domain with an I-CreI monomer (Chevalier,
Kortemme et al. 2002; Epinat, Arnould et al. 2003); International
PCT Application WO 03/078619 (Cellectis) and WO 2004/031346 (Fred
Hutchinson Cancer Research Center, Stoddard et al)) have
demonstrated the plasticity of LAGLIDADG proteins.
[0238] Different groups have also used a semi-rational approach to
locally alter the specificity of the I-CreI (Seligman, Stephens et
al. 1997; Sussman, Chadsey et al. 2004); International PCT
Applications WO 2006/097784, WO 2006/097853, WO 2007/060495 and WO
2007/049156 (Cellectis); (Arnould, Chames et al. 2006; Rosen,
Morrison et al. 2006; Smith, Grizot et al. 2006), I-SceI (Doyon,
Pattanayak et al. 2006), PI-SceI (Gimble, Moure et al. 2003) and
I-MsoI (Ashworth, Havranek et al. 2006).
[0239] In addition, hundreds of I-CreI derivatives with locally
altered specificity were engineered by combining the semi-rational
approach and High Throughput Screening: [0240] Residues Q44, R68
and R70 or Q44, R68, D75 and 177 of I-CreI were mutagenized and a
collection of variants with altered specificity at positions.+-.3
to 5 of the DNA target (5NNN DNA target) were identified by
screening (International PCT Applications WO 2006/097784 and WO
2006/097853 (Cellectis); (Arnould, Chames et al. 2006; Smith,
Grizot et al. 2006). [0241] Residues K28, N30 and Q38 or N30, Y33
and Q38 or K28, Y33, Q38 and S40 of I-CreI were mutagenized and a
collection of variants with altered specificity at positions .+-.8
to 10 of the DNA target (10NNN DNA target) were identified by
screening (Arnould, Chames et al. 2006; Smith, Grizot et al. 2006);
International PCT Applications WO 2007/060495 and WO 2007/049156
(Cellectis)).
[0242] Two different variants were combined and assembled in a
functional heterodimeric endonuclease able to cleave a chimeric
target resulting from the fusion of two different halves of each
variant DNA target sequence ((Arnould, Chames et al. 2006; Smith,
Grizot et al. 2006); International PCT Applications WO 2006/097854
and WO 2007/034262).
[0243] Furthermore, residues 28 to 40 and 44 to 77 of I-CreI were
shown to form two partially separable functional subdomains, able
to bind distinct parts of a homing endonuclease target half-site
(Smith, Grizot et al. 2006); International PCT Applications WO
2007/049095 and WO 2007/057781 (Cellectis)).
[0244] The combination of mutations from the two subdomains of
I-CreI within the same monomer allowed the design of novel chimeric
molecules (homodimers) able to cleave a palindromic combined DNA
target sequence comprising the nucleotides at positions .+-.3 to 5
and .+-.8 to 10 which are bound by each subdomain ((Smith, Grizot
et al. 2006); International PCT Applications WO 2007/049095 and WO
2007/057781 (Cellectis)).
[0245] The method for producing meganuclease variants and the
assays based on cleavage-induced recombination in mammal or yeast
cells, which are used for screening variants with altered
specificity are described in the International PCT Application WO
2004/067736; (Epinat, Arnould et al. 2003; Chames, Epinat et al.
2005; Arnould, Chames et al. 2006). These assays result in a
functional LacZ reporter gene which can be monitored by standard
methods.
[0246] The combination of the two former steps allows a larger
combinatorial approach, involving four different subdomains. The
different subdomains can be modified separately and combined to
obtain an entirely redesigned meganuclease variant (heterodimer or
single-chain molecule) with chosen specificity. In a first step,
couples of novel meganucleases are combined in new molecules
("half-meganucleases") cleaving palindromic targets derived from
the target one wants to cleave. Then, the combination of such
"half-meganucleases" can result in a heterodimeric species cleaving
the target of interest. The assembly of four sets of mutations into
heterodimeric endonucleases cleaving a model target sequence or a
sequence from different genes has been described in the following
Cellectis International patent applications: XPC gene
(WO2007/093918), RAG gene (WO2008/010093), HPRT gene
(WO2008/059382), beta-2 microglobulin gene (WO2008/102274), Rosa26
gene (WO2008/152523), Human hemoglobin beta gene (WO2009/13622) and
Human interleukin-2 receptor gamma chain gene (WO2009019614).
[0247] These variants can be used to cleave genuine chromosomal
sequences and have paved the way for novel perspectives in several
fields, including gene therapy.
[0248] Examples of such endonuclease include I-Sce I, I-Chu I,
I-Cre I, I-Csm I, PI-Sce I, PI-Tli I, PI-Mtu I, I-Ceu I, I-Sce II,
I-Sce III, HO, PI-Civ I, PI-Ctr I, PI-Aae I, PI-Bsu I, PI-Dha I,
PI-Dra I, PI-May I, PI-Mch I, PI-Mfu PI-MfI I, PI-Mga I, PI-Mgo I,
PI-Min I, PI-Mka I, PI-Mle I, PI-Mma I, PI-Msh I, PI-Msm I, PI-Mth
I, PI-Mtu I, PI-Mxe I, PI-Npu I, PI-Pfu I, PI-Rma I, PI-Spb I,
PI-Ssp I, PI-Fac I, PI-Mja I, PI-Pho I, PI-Tag I, PI-Thy I, PI-Tko
I, PI-Tsp I, I-MsoI.
[0249] A homing endonuclease can be a LAGLIDADG endonuclease such
as I-SceI, I-CreI, I-CeuI, I-MsoI, and I-DmoI.
[0250] Said LAGLIDADG endonuclease can be I-Sce I, a member of the
family that contains two LAGLIDADG motifs and functions as a
monomer, its molecular mass being approximately twice the mass of
other family members like I-CreI which contains only one LAGLIDADG
motif and functions as homodimers.
[0251] Endonucleases mentioned in the present application encompass
both wild-type (naturally-occurring) and variant endonucleases.
Endonucleases according to the invention can be a "variant"
endonuclease, i.e. an endonuclease that does not naturally exist in
nature and that is obtained by genetic engineering or by random
mutagenesis, i.e. an engineered endonuclease. This variant
endonuclease can for example be obtained by substitution of at
least one residue in the amino acid sequence of a wild-type,
naturally-occurring, endonuclease with a different amino acid. Said
substitution(s) can for example be introduced by site-directed
mutagenesis and/or by random mutagenesis. In the frame of the
present invention, such variant endonucleases remain functional,
i.e. they retain the capacity of recognizing (binding function) and
optionally specifically cleaving a target sequence to initiate gene
targeting process.
[0252] The variant endonuclease according to the invention cleaves
a target sequence that is different from the target sequence of the
corresponding wild-type endonuclease. Methods for obtaining such
variant endonucleases with novel specificities are well-known in
the art.
[0253] Endonucleases variants may be homodimers (meganuclease
comprising two identical monomers) or heterodimers (meganuclease
comprising two non-identical monomers). It is understood that the
scope of the present invention also encompasses endonuclease
variants per se, including heterodimers (WO2006097854), obligate
heterodimers (WO2008093249) and single chain meganucleases
(WO03078619 and WO2009095793) as non limiting examples, able to
cleave one target of interest in a polynucleotidic sequence or in a
genome. The invention also encompasses hybrid variant per se
composed of two monomers from different origins (WO03078619).
[0254] Endonucleases with novel specificities can be used in the
method according to the present invention for gene targeting and
thereby integrating a transgene of interest into a genome at a
predetermined location. [0255] by "parent meganuclease" it is
intended to mean a wild type meganuclease or a variant of such a
wild type meganuclease with identical properties or alternatively a
meganuclease with some altered characteristics in comparison to a
wild type version of the same meganuclease. This expression can
also be transposed to an endonuclease, a rare-cutting endonuclease,
a chimeric rare-cutting endonuclease, a TALEN or a compact TALEN
and derivatives. [0256] By "delivery vector" or "delivery vectors"
is intended any delivery vector which can be used in the present
invention to put into cell contact (i.e. "contacting") or deliver
inside cells or subcellular compartments agents/chemicals and
molecules (proteins or nucleic acids) needed in the present
invention. It includes, but is not limited to liposomal delivery
vectors, viral delivery vectors, drug delivery vectors, chemical
carriers, polymeric carriers, lipoplexes, polyplexes, dendrimers,
microbubbles (ultrasound contrast agents), nanoparticles, emulsions
or other appropriate transfer vectors. These delivery vectors allow
delivery of molecules, chemicals, macromolecules (genes, proteins),
or other vectors such as plasmids, peptides developed by Diatos. In
these cases, delivery vectors are molecule carriers. By "delivery
vector" or "delivery vectors" is also intended delivery methods to
perform transfection. [0257] The terms "vector" or "vectors" refer
to a nucleic acid molecule capable of transporting another nucleic
acid to which it has been linked. A "vector" in the present
invention includes, but is not limited to, a viral vector, a
plasmid, a RNA vector or a linear or circular DNA or RNA molecule
which may consists of a chromosomal, non chromosomal,
semi-synthetic or synthetic nucleic acids. Preferred vectors are
those capable of autonomous replication (episomal vector) and/or
expression of nucleic acids to which they are linked (expression
vectors). Large numbers of suitable vectors are known to those of
skill in the art and commercially available.
[0258] Viral vectors include retrovirus, adenovirus, parvovirus
(e.g. adenoassociated viruses), coronavirus, negative strand RNA
viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus
(e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g.
measles and Sendai), positive strand RNA viruses such as
picornavirus and alphavirus, and double-stranded DNA viruses
including adenovirus, herpesvirus (e.g., Herpes Simplex virus types
1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g.,
vaccinia, fowlpox and canarypox). Other viruses include Norwalk
virus, togavirus, flavivirus, reoviruses, papovavirus,
hepadnavirus, and hepatitis virus, for example. Examples of
retroviruses include: avian leukosis-sarcoma, mammalian C-type,
B-type viruses, D type viruses, HTLV-BLV group, lentivirus,
spumavirus (Coffin, J. M., Retroviridae: The viruses and their
replication, In Fundamental Virology, Third Edition, B. N. Fields,
et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).
[0259] By "lentiviral vector" is meant HIV-Based lentiviral vectors
that are very promising for gene delivery because of their
relatively large packaging capacity, reduced immunogenicity and
their ability to stably transduce with high efficiency a large
range of different cell types. Lentiviral vectors are usually
generated following transient transfection of three (packaging,
envelope and transfer) or more plasmids into producer cells. Like
HIV, lentiviral vectors enter the target cell through the
interaction of viral surface glycoproteins with receptors on the
cell surface. On entry, the viral RNA undergoes reverse
transcription, which is mediated by the viral reverse transcriptase
complex. The product of reverse transcription is a double-stranded
linear viral DNA, which is the substrate for viral integration in
the DNA of infected cells. [0260] By "integrative lentiviral
vectors (or LV)", is meant such vectors as non limiting example,
that are able to integrate the genome of a target cell. [0261] At
the opposite by "non integrative lentiviral vectors (or NILV)" is
meant efficient gene delivery vectors that do not integrate the
genome of a target cell through the action of the virus
integrase.
[0262] One type of preferred vector is an episome, i.e., a nucleic
acid capable of extra-chromosomal replication. Preferred vectors
are those capable of autonomous replication and/or expression of
nucleic acids to which they are linked. Vectors capable of
directing the expression of genes to which they are operatively
linked are referred to herein as "expression vectors. A vector
according to the present invention comprises, but is not limited
to, a YAC (yeast artificial chromosome), a BAC (bacterial
artificial), a baculovirus vector, a phage, a phagemid, a cosmid, a
viral vector, a plasmid, a RNA vector or a linear or circular DNA
or RNA molecule which may consist of chromosomal, non chromosomal,
semi-synthetic or synthetic DNA. In general, expression vectors of
utility in recombinant DNA techniques are often in the form of
"plasmids" which refer generally to circular double stranded DNA
loops which, in their vector form are not bound to the chromosome.
Large numbers of suitable vectors are known to those of skill in
the art. Vectors can comprise selectable markers, for example:
neomycin phosphotransferase, histidinol dehydrogenase,
dihydrofolate reductase, hygromycin phosphotransferase, herpes
simplex virus thymidine kinase, adenosine deaminase, glutamine
synthetase, and hypoxanthine-guanine phosphoribosyl transferase for
eukaryotic cell culture; TRP1 for S. cerevisiae; tetracyclin,
rifampicin or ampicillin resistance in E. coli. Preferably said
vectors are expression vectors, wherein a sequence encoding a
polypeptide of interest is placed under control of appropriate
transcriptional and translational control elements to permit
production or synthesis of said polypeptide. Therefore, said
polynucleotide is comprised in an expression cassette. More
particularly, the vector comprises a replication origin, a promoter
operatively linked to said encoding polynucleotide, a ribosome
binding site, a RNA-splicing site (when genomic DNA is used), a
polyadenylation site and a transcription termination site. It also
can comprise an enhancer or silencer elements. Selection of the
promoter will depend upon the cell in which the polypeptide is
expressed. Suitable promoters include tissue specific and/or
inducible promoters. Examples of inducible promoters are:
eukaryotic metallothionine promoter which is induced by increased
levels of heavy metals, prokaryotic lacZ promoter which is induced
in response to isopropyl-.beta.-D-thiogalacto-pyranoside (IPTG) and
eukaryotic heat shock promoter which is induced by increased
temperature. Examples of tissue specific promoters are skeletal
muscle creatine kinase, prostate-specific antigen (PSA),
.alpha.-antitrypsin protease, human surfactant (SP) A and B
proteins, .beta.-casein and acidic whey protein genes.
[0263] Inducible promoters may be induced by pathogens or stress,
more preferably by stress like cold, heat, UV light, or high ionic
concentrations (reviewed in Potenza C et al. 2004, In vitro Cell
Dev Biol 40:1-22). Inducible promoter may be induced by chemicals
(reviewed in (Moore, Samalova et al. 2006); (Padidam 2003); (Wang,
Zhou et al. 2003); (Zuo and Chua 2000).
[0264] Delivery vectors and vectors can be associated or combined
with any cellular permeabilization techniques such as sonoporation
or electroporation or derivatives of these techniques. [0265] By
cell or cells is intended any prokaryotic or eukaryotic living
cells, cell lines derived from these organisms for in vitro
cultures, primary cells from animal or plant origin. [0266] By
"primary cell" or "primary cells" are intended cells taken directly
from living tissue (i.e. biopsy material) and established for
growth in vitro, that have undergone very few population doublings
and are therefore more representative of the main functional
components and characteristics of tissues from which they are
derived from, in comparison to continuous tumorigenic or
artificially immortalized cell lines. These cells thus represent a
more valuable model to the in vivo state they refer to. [0267] In
the frame of the present invention, "eukaryotic cells" refer to a
fungal, plant or animal cell or a cell line derived from the
organisms listed below and established for in vitro culture. More
preferably, the fungus is of the genus Aspergillus, Penicillium,
Acremonium, Trichoderma, Chrysoporium, Mortierella, Kluyveromyces
or Pichia; More preferably, the fungus is of the species
Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae,
Aspergillus terreus, Penicillium chrysogenum, Penicillium citrinum,
Acremonium Chrysogenum, Trichoderma reesei, Mortierella alpine,
Chrysosporium lucknowense, Kluyveromyces lactis, Pichia pastoris or
Pichia ciferrii.
[0268] More preferably the plant is of the genus Arabidospis,
Nicotiana, Solanum, lactuca, Brassica, Oryza, Asparagus, Pisum,
Medicago, Zea, Hordeum, Secale, Triticum, Capsicum, Cucumis,
Cucurbita, Citrullis, Citrus, Sorghum; More preferably, the plant
is of the species Arabidospis thaliana, Nicotiana tabaccum, Solanum
lycopersicum, Solanum tuberosum, Solanum melongena, Solanum
esculentum, Lactuca saliva, Brassica napus, Brassica oleracea,
Brassica rapa, Oryza glaberrima, Oryza sativa, Asparagus
officinalis, Pisum sativum, Medicago sativa, zea mays, Hordeum
vulgare, Secale cereal, Triticum aestivum, Triticum durum, Capsicum
sativus, Cucurbita pepo, Citrullus lanatus, Cucumis melo, Citrus
aurantifolia, Citrus maxima, Citrus medica, Citrus reticulata.
[0269] More preferably the animal cell is of the genus Homo,
Rattus, Mus, Sus, Bos, Danio, Canis, Felis, Equus, Salmo,
Oncorhynchus, Gallus, Meleagris, Drosophila, Caenorhabditis; more
preferably, the animal cell is of the species Homo sapiens, Rattus
norvegicus, Mus musculus, Sus scrofa, Bos taurus, Danio rerio,
Canis lupus, Felis catus, Equus caballus, Salmo salar, Oncorhynchus
mykiss, Gallus gallus, Meleagris gallopavo, Drosophila
melanogaster, Caenorhabditis elegans.
[0270] In the present invention, the cell can be a plant cell, a
mammalian cell, a fish cell, an insect cell or cell lines derived
from these organisms for in vitro cultures or primary cells taken
directly from living tissue and established for in vitro culture.
As non-limiting examples, cell can be protoplasts obtained from
plant organisms listed above. As non limiting examples cell lines
can be selected from the group consisting of CHO-K1 cells; HEK293
cells; Caco2 cells; U2-OS cells; NIH 3T3 cells; NSO cells; SP2
cells; CHO-S cells; DG44 cells; K-562 cells, U-937 cells; MRCS
cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLa cells; HT-1080
cells; HCT-116 cells; Hu-h7 cells; Huvec cells; Molt 4 cells.
[0271] All these cell lines can be modified by the method of the
present invention to provide cell line models to produce, express,
quantify, detect, study a gene or a protein of interest; these
models can also be used to screen biologically active molecules of
interest in research and production and various fields such as
chemical, biofuels, therapeutics and agronomy as non-limiting
examples. Adoptive immunotherapy using genetically engineered T
cells is a promising approach for the treatment of malignancies and
infectious diseases. Most current approaches rely on gene transfer
by random integration of an appropriate T Cell Receptor (TCR) or
Chimeric Antigen Receptor (CAR). Targeted approach using
rare-cutting endonucleases is an efficient and safe alternative
method to transfer genes into T cells and generate genetically
engineered T cells. [0272] by "homologous" is intended a sequence
with enough identity to another one to lead to homologous
recombination between sequences, more particularly having at least
95% identity, preferably 97% identity and more preferably 99%.
[0273] identity" refers to sequence identity between two nucleic
acid molecules or polypeptides. Identity can be determined by
comparing a position in each sequence which may be aligned for
purposes of comparison. When a position in the compared sequence is
occupied by the same base, then the molecules are identical at that
position. A degree of similarity or identity between nucleic acid
or amino acid sequences is a function of the number of identical or
matching nucleotides at positions shared by the nucleic acid
sequences. Various alignment algorithms and/or programs may be used
to calculate the identity between two sequences, including FASTA,
or BLAST which are available as a part of the GCG sequence analysis
package (University of Wisconsin, Madison, Wis.), and can be used
with, e.g., default setting. [0274] by "mutation" is intended the
substitution, deletion, insertion of one or more nucleotides/amino
acids in a polynucleotide (cDNA, gene) or a polypeptide sequence.
Said mutation can affect the coding sequence of a gene or its
regulatory sequence. It may also affect the structure of the
genomic sequence or the structure/stability of the encoded mRNA.
[0275] In the frame of the present invention, the expression
"double-strand break-induced mutagenesis" (DSB-induced mutagenesis)
refers to a mutagenesis event consecutive to an NHEJ event
following an endonuclease-induced DSB, leading to
insertion/deletion at the cleavage site of an endonuclease. [0276]
By "gene" is meant the basic unit of heredity, consisting of a
segment of DNA arranged in a linear manner along a chromosome,
which codes for a specific protein or segment of protein. A gene
typically includes a promoter, a 5' untranslated region, one or
more coding sequences (exons), optionally introns, a 3'
untranslated region. The gene may further comprise a terminator,
enhancers and/or silencers. [0277] As used herein, the term
"transgene" refers to a sequence encoding a polypeptide.
Preferably, the polypeptide encoded by the transgene is either not
expressed, or expressed but not biologically active, in the cell,
tissue or individual in which the transgene is inserted. Most
preferably, the transgene encodes a therapeutic polypeptide useful
for the treatment of an individual. [0278] The term "gene of
interest" or "GOI" refers to any nucleotide sequence encoding a
known or putative gene product. [0279] As used herein, the term
"locus" is the specific physical location of a DNA sequence (e.g.
of a gene) on a chromosome. The term "locus" usually refers to the
specific physical location of an endonuclease's target sequence on
a chromosome. Such a locus, which comprises a target sequence that
is recognized and cleaved by an endonuclease according to the
invention, is referred to as "locus according to the invention".
Also, the expression "genomic locus of interest" is used to qualify
a nucleic acid sequence in a genome that can be a putative target
for a double-strand break according to the invention. It is
understood that the considered genomic locus of interest of the
present invention can not only qualify a nucleic acid sequence that
exists in the main body of genetic material (i.e. in a chromosome)
of a cell but also a portion of genetic material that can exist
independently to said main body of genetic material such as
plasmids, episomes, virus, transposons or in organelles such as
mitochondria or chloroplasts as non-limiting examples. [0280] By
the expression "loss of genetic information" is understood the
elimination or addition of at least one given DNA fragment (at
least one nucleotide) or sequence, bordering the recognition sites
of the endonucleases, chimeric rare-cutting endonucleases, compact
TALEN or enhanced compact TALEN of the present invention or the
intervening sequence between at least two processing sites of the
endonucleases, chimeric rare-cutting endonucleases, compact TALEN
or enhanced compact TALEN of the present invention and leading to a
change of the original sequence around said endonuclease-cutting
sites, chimeric rare-cutting endonuclease-cutting sites, compact
TALEN or enhanced compact TALEN recognition DNA binding site within
the genomic locus of interest. This loss of genetic information can
be, as a non-limiting example, the elimination of an intervening
sequence between two endonuclease-cutting sitesor between two
processing sites of a compact TALEN or enhanced compact TALEN. As
another non-limiting example, this loss of genetic information can
also be an excision of a single-strand of DNA spanning the binding
region of a compact TALEN or an enhanced compact TALEN according to
the present invention [0281] By "scarless re-ligation" or "scarless
religation" is intended the perfect re-ligation event, without loss
of genetic information (no insertion/deletion events) of the DNA
broken ends through NHEJ process after the creation of a
double-strand break event. [0282] By "Imprecise NHEJ" is intended
the re-ligation of nucleic acid ends generated by a DSB, with
insertions or deletions of nucleotides. Imprecise NHEJ is an
outcome and not a repair pathway and can result from different NHEJ
pathways (Ku dependent or Ku independent as non-limiting examples).
[0283] By "fusion protein" is intended the result of a well-known
process in the art consisting in the joining of two or more genes
which originally encode for separate proteins or part of them, the
translation of said "fusion gene" resulting in a single polypeptide
with functional properties derived from each of the original
proteins. [0284] By "chimeric rare-cutting endonuclease" is meant
any fusion protein comprising a rare-cutting endonuclease. Said
rare-cutting endonuclease might be at the N-terminal part of said
chimeric rare-cutting endonuclease; at the opposite, said
rare-cutting endonuclease might be at the C-terminal part of said
chimeric rare-cutting endonuclease. A "chimeric rare-cutting
endonuclease" according to the present invention which comprises
two catalytic domains can be described as "bi-functional" or as
"bi-functional meganuclease". A "chimeric rare-cutting
endonuclease" according to the present invention which comprises
more than two catalytic domains can be described as
"multi-functional" or as "multi-functional meganuclease". As
non-limiting examples, chimeric rare-cutting endonucleases
according to the present invention can be a fusion protein between
a rare-cutting endonuclease and one catalytic domain; chimeric
rare-cutting endonucleases according to the present invention can
also be a fusion protein between a rare-cutting endonuclease and
two catalytic domains. As mentioned previously, the rare-cutting
endonuclease part of chimeric rare-cutting endonucleases according
to the present invention can be a meganuclease comprising two
identical monomers, two non-identical monomers, or a single chain
meganuclease. The rare-cutting endonuclease part of chimeric
rare-cutting endonucleases according to the present invention can
also be the DNA-binding domain of an inactive rare-cutting
endonuclease. In other non-limiting examples, chimeric rare-cutting
endonucleases according to the present invention can be derived
from a TALE-nuclease (TALEN), i.e. a fusion between a DNA-binding
domain derived from a Transcription Activator Like Effector (TALE)
and one or two catalytic domains. In other non-limiting examples, a
subclass of chimeric rare-cutting endonucleases according to the
present invention can be a "compact TALE-nuclease" (cTALEN), i.e. a
fusion between an engineered core TALE scaffold comprising at least
a Repeat Variable Dipeptide regions domain and at least one
catalytic domain, said fusion protein constituting a compact TALEN
active entity that does not require dimerization for DNA processing
activity. Said catalytic domain can be an endonuclease as listed in
table 2 as non-limiting examples; said catalytic domain can be a
frequent-cutting endonuclease such as a restriction enzyme selected
from the group consisting of MmeI, R-HinPII, R.MspI, R.MvaI,
Nb.BsrDI, BsrDI A, Nt.BspD6I, ss.BspD6I, R.PleI, MlyI and AlwI as
non-limiting restriction enzymes examples listed in table 2. [0285]
By "enhancer domain(s)" or "enhancer(s)" are meant protein domains
that provide functional and/or structural support to a protein
scaffold, a compact TALEN as a non-limiting example, therefore
allowing an enhancement in global DNA processing efficiency of the
resulting fusion protein, i.e. an enhanced compact TALEN, relative
to the DNA processing efficiency of the starting compact TALEN. A
particular domain is an enhancer domain when it provides at least a
5% enhancement in efficiency of the starting scaffold, more
preferably 10%, again more preferably 15%, again more preferably
20%, again more preferably 25%, again more preferably 50%, again
more preferably greater than 50%. Non-limiting examples of such
enhancer domains are given in Tables 1 and 2. By "auxiliary
enhancer domains" or "auxiliary enhancers" or "auxiliary domains"
are meant protein domains acting in trans with a compact TALEN or
an enhanced compact TALEN to provide an additional function that is
not essential for said basic compact TALEN activity or said
enhanced compact TALEN activity. When such auxiliary enhancers are
used, compact TALEN or enhanced compact TALEN are converted to
"trans TALEN", respectively trans compact TALEN and trans enhanced
compact TALEN. [0286] By "catalytic domain" is intended the protein
domain or module of an enzyme containing the active site of said
enzyme; by active site is intended the part of said enzyme at which
catalysis of the substrate occurs. Enzymes, but also their
catalytic domains, are classified and named according to the
reaction they catalyze. The Enzyme Commission number (EC number) is
a numerical classification scheme for enzymes, based on the
chemical reactions they catalyze
(http://www.chem.qmul.ac.ukhubmb/enzyme/). In the scope of the
present invention, any catalytic domain can be fused to an
engineered core TALE scaffold to generate a compact TALEN active
entity with a DNA processing efficiency provided by at least said
catalytic domain activity. Said catalytic domain can provide a
nuclease activity (endonuclease or exonuclease activities, cleavase
or nickase), a polymerase activity, a kinase activity, a
phosphatase activity, a methylase activity, a topoisomerase
activity, an integrase activity, a transposase activity or a ligase
activity as non-limiting examples. Non-limiting examples of such
catalytic domains are given in Tables 1 and 2. In a preferred
embodiment of the present invention, said catalytic domain can be
considered as an enhancer domain. If catalytically active, said
enhancer domain can provide functional and/or structural support to
the compact TALEN scaffold leading to an enhanced compact TALEN
when fused to it. If catalytically inactive, said enhancer domain
provides structural support to compact TALEN scaffold leading to an
enhanced compact TALEN when fused to it. It can be envisioned from
the present invention to fuse catalytic domains according to the
present invention to one part of a classical TALEN in order to give
these classical TALENs new catalytical properties provided by at
least said catalytic domain activity or to improve their DNA
processing efficiency. [0287] By "nuclease catalytic domain" is
intended the protein domain comprising the active site of an
endonuclease or an exonuclease enzyme. Such nuclease catalytic
domain can be, for instance, a "cleavase domain" or a "nickase
domain". By "cleavase domain" is intended a protein domain whose
catalytic activity generates a Double Strand Break (DSB) in a DNA
target. By "nickase domain" is intended a protein domain whose
catalytic activity generates a single strand break in a DNA target
sequence. Non-limiting examples of such catalytic domains are given
in Tables 1 and 2 with a GenBank or NCBI or UniProtKB/Swiss-Prot
number as a reference. [0288] By a "TALE-nuclease" (TALEN) or a
"classical TALEN" is intended a fusion protein consisting of a
DNA-binding domain derived from a Transcription Activator Like
Effector (TALE) and one FokI catalytic domain, that need to
dimerize to form an active entity able to cleave a DNA target
sequence. [0289] By "compact TALE-nuclease" (cTALEN) is intended a
general designation according to the present invention for a fusion
protein between an engineered core TALE scaffold comprising at
least one Repeat Variable Dipeptides domain and at least one
catalytic domain, said fusion protein constituting a compact TALEN
(or cTALEN) active entity and not requiring dimerization for DNA
processing activity. Compact TALENs are designed to alleviate the
need for multiple independent protein moieties when targeting a DNA
cleavage event. Importantly, the requisite "spacer" region and dual
target sites essential for the function of current TALENs are
unnecessary. In other words, the compact TALEN according to the
present invention is an active entity unit able, by itself, to
target only one specific single double-stranded DNA target sequence
of interest through one DNA binding domain and to process DNA
nearby said single double-stranded DNA target sequence of interest.
In addition, since the catalytic domain does not require specific
DNA contact, there are no restrictions on regions surrounding the
core TALE DNA binding domain. In the scope of the present
invention, it can be also envisioned some sequence preference in
the catalytic domain. When a cTALEN comprises only one catalytic
domain, cTALEN can be qualified as a "basic cTALEN" or "cTALEN".
When a cTALEN further comprises at least one "enhancer domain",
cTALEN can be qualified as an enhanced cTALEN or an "eTALEN". A
cTALEN or an eTALEN that comprise at least one cleavase catalytic
domain and one nickase catalytic domain or at least two cleavase
catalytic domains can be specifically qualified as a dual-cleavage
cTALEN or a "dcTALEN". A cTALEN or an eTALEN acting with an
auxiliary domain in trans is qualified as a trans compact TALEN or
a trans enhanced compact TALEN, both being "trans TALEN".
[0290] The above written description of the invention provides a
manner and process of making and using it such that any person
skilled in this art is enabled to make and use the same, this
enablement being provided in particular for the subject matter of
the appended claims, which make up a part of the original
description.
[0291] As used above, the phrases "selected from the group
consisting of," "chosen from," and the like include mixtures of the
specified materials.
[0292] Where a numerical limit or range is stated herein, the
endpoints are included. Also, all values and subranges within a
numerical limit or range are specifically included as if explicitly
written out.
[0293] The above description is presented to enable a person
skilled in the art to make and use the invention, and is provided
in the context of a particular application and its requirements.
Various modifications to the preferred embodiments will be readily
apparent to those skilled in the art, and the generic principles
defined herein may be applied to other embodiments and applications
without departing from the spirit and scope of the invention. Thus,
this invention is not intended to be limited to the embodiments
shown, but is to be accorded the widest scope consistent with the
principles and features disclosed herein.
[0294] Having generally described this invention, a further
understanding can be obtained by reference to certain specific
examples, which are provided herein for purposes of illustration
only, and are not intended to be limiting unless otherwise
specified.
EXAMPLES
Example 1
[0295] The wild-type I-CreI meganuclease (SEQ ID NO: 106) was
chosen as the parent scaffold on which to fuse the catalytic domain
of I-TevI (SEQ ID NO: 107). Wild-type I-TevI functions as a
monomeric cleavase of the GIY-YIG family to generate a staggered
double-strand break in its target DNA. Guided by biochemical and
structural data, variable length constructs were designed from the
N-terminal region of 1-TevI that encompass the entire catalytic
domain and deletion-intolerant region of its linker (SEQ ID NO: 109
to SEQ ID NO: 114). In all but one case, fragments were fused to
the N-terminus of I-CreI with an intervening 5-residue polypeptide
linker (-QGPSG-; SEQ ID NO: 103). The linker-less fusion construct
naturally contained residues (-LGPDGRKA-; SEQ ID NO: 104) similar
to those in the artificial linker. As I-CreI is a homodimer, all
fusion constructs contain three catalytic centers (FIG. 4, where
"catalytic domain"=cleavase): the natural I-CreI active site at the
interface of the dimer and one I-TevI active site per monomer.
[0296] The activity of each "tri-functional" meganuclease was
assessed using our yeast assay previously described in
International PCT Applications WO 2004/067736 and in (Epinat,
Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et
al. 2006; Smith, Grizot et al. 2006). All constructs were able to
cleave the C1221 target DNA with an activity comparable to that of
wild-type I-CreI (Table 4). To validate the activity of the I-TevI
catalytic domain independent of the I-CreI catalytic core, D20N
point mutants were made to inactivate the I-CreI scaffold [SEQ ID
NO: 108, SEQ ID NO: 115 to SEQ ID NO: 120; Chevalier, Sussman et
al. 2004)]. Tests in our yeast assays showed no visible activity
from the inactivated I-CreI (D20N) mutant protein alone (Table 4).
However, cleavage activity could be observed for fusions having the
I-TevI catalytic domain (Table 4).
TABLE-US-00005 TABLE 4 Activity in Yeast assay for I-TevI/I-CreI
fusions. The relative activity of wild-type and fusion proteins on
the two parent protein targets (C1221 for I-CreI and Tev for
I-TevI) is shown. Maximal activity (++++) is seen with each given
protein on its native DNA target. I-CreI_N20 is an inactive variant
of the wild-type I-CreI scaffold. In all other cases, activity is
only detected on the C1221 target since DNA recognition is driven
by the I-CreI scaffold. The "N20" fusion variants illustrate
cleavage activity due to the I-TevI catalytic domain. Relative
Activity in Yeast Assay (37.degree. C.) Protein Construct C1221
Target Tev Target I-CreI ++++ - I-TevI - ++++ I-CreI_N20 - -
hTevCre_D01 ++++ - hTevCre_D02 ++++ - hTevCre_D03 ++++ -
hTevCre_D04 ++++ - hTevCre_D05 ++++ - hTevCre_D06 ++++ -
hTevCre_D01_N20 ++ - hTevCre_D02_N20 ++ - hTevCre_D03_N20 ++ -
hTevCre_D04_N20 ++ - hTevCre_D05_N20 - - hTevCre_D06_N20 - -
Relative activity is scaled as: -, no activity detectable; +,
<25% activity; ++, 25% to <50% activity; +++, 50% to <75%
activity; ++++, 75% to 100% activity.
Example 2
[0297] Protein-fusion scaffolds were designed based on a truncated
form of I-CreI (SEQ ID NO: 106, I-CreI_X: SEQ ID NO: 121) and three
different linker polypeptides (NFS1=SEQ ID NO: 98; NFS2=SEQ ID NO:
99; CFS1=SEQ ID NO: 100) fused to either the N- or C-terminus of
the protein. Structure models were generated in all cases, with the
goal of designing a "baseline" fusion linker that would traverse
the I-CreI parent scaffold surface with little to no effect on its
DNA binding or cleavage activities. For the two N-terminal fusion
scaffolds, the polypeptide spanning residues 2 to 153 of I-CreI was
used, with a K82A mutation to allow for linker placement. The
C-terminal fusion scaffold contains residues 2 to 155 of wild-type
I-CreI. For both fusion scaffold types, the "free" end of the
linker (i.e. onto which a polypeptide can be linked) is designed to
be proximal to the DNA, as determined from models built using the
I-CreI/DNA complex structures as a starting point (PDB id: 1g9z).
The two I-CreI N-terminal fusion scaffolds (1-Crel_NFS1=SEQ ID NO:
122 and I-CreI_NFS2=SEQ ID NO: 123) and the single C-terminal
fusion scaffold (1-Crel_CFS1=SEQ ID NO: 124) were tested in our
yeast assay (see Example 1) and found to have activity similar to
that of wild-type I-CreI (Table 5).
TABLE-US-00006 TABLE 5 Activity in Yeast assay for ColE7/I-CreI
fusions. The relative activity of wild-type and fusion proteins on
theC1221 target is shown. I-CreI_X represents a truncated version
of I-CreI based on the crystal structure and was used as the
foundation for the fusion scaffolds (I-CreI_NFS1, I-CreI_NFS2 and
I-CreI_CFS1). "N20" constructs are inactive variants of the
respective I-CreI-based scaffolds. Activity is detected in all
cases wherein the I-CreI scaffold is active or when DNA catalysis
is provided by the ColE7 domain. Relative Activity in Yeast Assay
(37.degree. C.) Protein Construct C1221 Target I-CreI ++++ I-CreI_X
++++ I-CreI_NFS1 ++++ I-CreI_NFS2 ++++ I-CreI_CFS1 ++++
I-CreI_NFS1_N20 - I-CreI_NFS2_N20 - I-CreI_CFS1_N20 -
hColE7Cre_D0101 ++++ hColE7Cre_D0102 ++++ hCreColE7_D0101 ++++
hColE7Cre_D0101_N20 +++ hColE7Cre_D0102_N20 +++ hCreColE7_D0101_N20
++ Relative activity is scaled as: -, no activity detectable; +,
<25% activity; ++, 25% to <50% activity; +++, 50% to <75%
activity; ++++, 75% to 100% activity.
[0298] Colicin E7 is a non-specific nuclease of the HNH family able
to process single- and double-stranded DNA (Hsia, Chak et al.
2004). Guided by biochemical and structural data, the region of
ColE7 that encompasses the entire catalytic domain (SEQ ID NO: 140;
(Hsia, Chak et al. 2004) was selected. This ColE7 domain was fused
to the N-terminus of either I-CreI_NFS1 (SEQ ID NO: 122) or
I-CreI_NFS2 (SEQ ID NO: 123) to create hColE7Cre_D0101 (SEQ ID NO:
128) or hColE7Cre_D0102 (SEQ ID NO: 129), respectively. In
addition, a C-terminal fusion construct, hCreColE7_D0101 (SEQ ID
NO: 130), was generated using I-CreI_CFS1 (SEQ ID NO: 124). As
I-CreI is a homodimer, all fusion constructs contain three
catalytic centers (FIG. 4, where "catalytic domain"=cleavage): the
natural I-CreI active site at the interface of the dimer and one
ColE7 active site per monomer.
[0299] The activity of each "tri-functional" meganuclease was
assessed using our yeast assay (see Example 1). All constructs were
able to cleave the C1221 target DNA with an activity comparable to
that of wild-type I-CreI (Table 5).
[0300] To validate the activity of the ColE7 catalytic domain
independent of the I-CreI catalytic core, D20N point mutants were
made to inactivate the I-CreI scaffold (SEQ ID NO: 125, SEQ ID NO:
126, SEQ ID NO: 127, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO:
133; (Chevalier, Sussman et al. 2004)). Tests in our yeast assays
showed no visible activity from the inactivated I-CreI (D20N)
mutant proteins alone (Table 5). However, cleavage activity could
be observed for fusions having the ColE7 catalytic domain (Table
5).
Example 3
[0301] Two core TALE scaffolds are generated onto which (a)
different sets of RVD domains could be inserted to change DNA
binding specificity, and; (b) a selection of catalytic domains
could be attached, N- or C-terminal, to effect DNA cleavage (or
nicking). The core scaffolds (sT1: SEQ ID NO: 134 and sT2: SEQ ID
NO: 135) differ in the N- and C-terminal regions, where sT2 is a
truncated variant lacking 152 amino acid residues from the
N-terminus (Szurek, Rossier et al. 2002) and the last 220 residues
from the C-terminus compared to sT1. In sT1, the C-terminal region
is a truncation with respect to wild-type TALE domains, ending at a
fortuitously defined restriction site (BamHI) in the DNA coding
sequence.
[0302] Using the two core scaffolds, four "baseline" TALE DNA
binding proteins (bT1-Avr=SEQ ID NO: 136, bT2-Avr=SEQ ID NO: 137,
bT1-Pth .dbd.SEQ ID NO 138 and bT2-Pth .dbd.SEQ ID NO 139) are
generated by insertion of the corresponding set of repeat domains
that recognize the naturally occurring asymmetric sequences AvrBs3
(19 bp) and PthXo1 (25 bp) (FIG. 3). Example protein sequences of
the baseline scaffolds are listed in SEQ ID NO: 136 to SEQ ID NO:
139. As is, these scaffolds can be tested in vitro for DNA binding
ability on targets having only a single recognition sequence. For
comparison with existing TALENs, the catalytic domain of the FokI
nuclease (SEQ ID NO: 368 and particularly residues P381 to F583 as
non-limiting example) can be fused to either the N- or C-terminus
of the baseline scaffolds. Effective cleavage using these controls
requires target site DNAs that contain two TALE binding
sequences.
[0303] In addition to verifying activity using naturally occurring
sequences, five artificial RVD constructs recognizing relevant
sequences were generated (FIG. 3): RagT2-R, NptIIT5-L, NptIIT5-R,
NptIIT6-L, NptIIT6-R. Example protein sequences of the insert RVDs
are listed in SEQ ID NO: 253 to SEQ ID NO: 257. Artificial RVD
sequences are used as noted above within the sT1 or sT2 scaffold to
generate the desired targeted compact TALENs.
[0304] Basic compact TALENs (cTALENs) are generated via fusion of
catalytic domains to either the N- or C-terminus of the baseline
scaffolds (FIG. 5, A or B, respectively). A non-exhaustive list of
catalytic domains amenable to fusion with TALE DNA binding domains
is presented in Table 2. A non-exhaustive list of linkers that can
be used is presented in Table 3. It is notable that linker design
can depend on the nature of the catalytic domain attached and its
given application. It can also be anticipated that specially
engineered linkers can be constructed to better control or regulate
the activity of either or both domains. Examples 5, 6 and 7 below
discuss additional and alternative methods in which linkers can be
defined. All cTALEN designs are assessed using our yeast assay (see
Example 1) and provide detectable activity comparable to existing
engineered meganucleases.
Example 3a
TALE::TevI Compact TALEN
[0305] The catalytic domain of I-TevI (SEQ ID NO: 20), a member of
the GIY-YIG endonuclease family, was fused to a TALE-derived
scaffold (composed of a N-terminal domain, a central core composed
of RVDs and a C-terminal domain) to create a new class of cTALEN
(TALE::TevI). To distinguish the orientation (N-terminal vs.
C-terminal) of the catalytic domain (CD) fusions, construct names
are written as either CD::TALE-RVD (catalytic domain is fused
N-terminal to the TALE domain) or TALE-RVD::CD (catalytic domain is
fused C-terminal to the TALE domain), where "-RVD" optionally
designates the sequence recognized by the TALE domain and "CD" is
the catalytic domain type. Herein, we describe novel TALE::TevI
constructions that target AvrBs3 sequence for example, thus named
TALE-AvrBs3::TevI.
[0306] Activity of TALE::TevI in Yeast
[0307] A core TALE scaffold, sT2 (SEQ ID NO: 135), was selected
onto which (a) different sets of RVD domains could be inserted to
change DNA binding specificity, and; (b) a selection of
I-TevI-derived catalytic domains could be attached, N- or
C-terminal, to effect DNA cleavage (or nicking). The previously
mentioned sT2 truncated scaffold was generated by the PCR from a
full-length core TALEN scaffold template (pCLS7183, SEQ ID NO: 141)
using primers CMP_G061 (SEQ ID NO: 142) and CMP_G065 (SEQ ID NO:
143) and was cloned into vector pCLS7865 (SEQ ID NO: 144) to
generate pCLS7865-cTAL11_CFS1 (pCLS9009, SEQ ID NO: 145), where
CFS1 designates the amino acid sequence -GSSG- (with underlying
restriction sites BamHI and Kpn21 in the coding DNA to facilitate
cloning). Three variants of the I-TevI (SEQ ID NO: 20) catalytic
domain were amplified by the PCR on templates TevCreD01 [SEQ ID NO:
109 protein in plasmid pCLS6614 (SEQ ID NO: 146)] using the primer
pair CMP_G069 (SEQ ID NO: 147) and CMP_G070 (SEQ ID NO: 148),
TevCreD02 [SEQ ID NO: 110 protein in plasmid pCLS6615 (SEQ ID NO:
203)] using the primer pair CMP_G069 (SEQ ID NO: 147) and CMP_G071
(SEQ ID NO: 149) or TevCreD05 [SEQ ID NO: 113 protein in plasmid
pCLS6618 (SEQ ID NO: 258)] using the primer pair CMP_G069 (SEQ ID
NO: 147) and CMP_G115 (SEQ ID NO: 259) and subcloned into the
pCLS9009 backbone by restriction and ligation using BamHI and EagI
restriction sites, yielding pCLS7865-cT11_TevD01 (pCLS9010, SEQ ID
NO: 150), pCLS7865-cT11_TevD02 (pCLS9011, SEQ ID NO: 151) and
pCLS7865-cT11_TevD05 (pCLS15775, SEQ ID NO: 260), respectively. All
fusions contain the dipeptide -GS- linking the TALE-derived DNA
binding domain and I-TevI-derived catalytic domain.
[0308] The DNA sequence coding for the RVDs to target the AvrBs3
site (SEQ ID NO: 152) was subcloned into both plasmids pCLS9010
(SEQ ID NO: 150, encoding the protein of SEQ ID NO: 420), pCLS9011
(SEQ ID NO: 151, encoding the protein of SEQ ID NO: 421) and
pCLS15775 (SEQ ID NO: 260, encoding the protein of SEQ ID NO: 422)
using Type IIS restriction enzymes BsmBI for the receiving plasmid
and BbvI and SfaNI for the inserted RVD sequence to create the
subsequent TALE-AvrBs3::TevI constructs cT11AvrTevD01 (pCLS9012,
SEQ ID NO: 218, encoding the protein of SEQ ID NO: 423),
cT11Avr_TevD02 (pCLS9013, SEQ ID NO: 153, encoding the protein of
SEQ ID NO: 424) and cT11Avr_TevD05 (pCLS15776, SEQ ID NO: 261,
encoding the protein of SEQ ID NO: 425), respectively. These
TALE-AvrBs3::TevI constructs were sequenced and the insert
transferred to additional vectors as needed (see below).
[0309] The final TALE-AvrBs3::TevI yeast expression plasmids,
pCLS8523 (SEQ ID NO: 154), pCLS8524 (SEQ ID NO: 155) and pCLS12092
(SEQ ID NO: 262), were prepared by yeast in vivo cloning using
plasmids pCLS9012, pCLS9013 and pCLS15776, respectively. To
generate an intact coding sequence by in vivo homologous
recombination, approximately 40 ng of each plasmid linearized by
digestion with BssHII and 1 ng of the pCLS0542 (SEQ ID NO: 156)
plasmid DNA linearized by digestion with NcoI and EagI were used to
transform, respectively, the yeast S. cerevisiae strain FYC2-6A
(MAT.alpha., trp1.DELTA.63, leu2.DELTA.1, his3.DELTA.200) using a
high efficiency LiAc transformation protocol (Arnould et al.
2007).
[0310] All the yeast target reporter plasmids containing the TALEN
DNA target sequences were constructed as previously described
(International PCT Applications WO 2004/067736 and in Epinat,
Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et
al. 2006; Smith, Grizot et al. 2006).
[0311] The TALE-AvrBs3::TevI constructs were tested in a yeast SSA
assay as previously described (International PCT Applications WO
2004/067736 and in Epinat, Arnould et al. 2003; Chames, Epinat et
al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al. 2006)
on pseudo palindromic targets in order to compare activity with a
standard TALE-AvrBs3::FokI TALEN (pCLS8590, SEQ ID NO: 244), which
requires two binding sites for activity. AvrBs3 targets contain two
identical recognition sequences juxtaposed with the 3' ends
proximal and separated by "spacer" DNA ranging from 5 to 40 bps
(SEQ ID NO: 157 to 192, Table 6). TALE-AvrBs3::TevI activity levels
on their respective targets in yeast cells are shown on FIG. 9.
Data summarized in FIG. 9 show that TALE-AvrBs3::TevI is active
against several targets in Yeast.
[0312] Activity of TALE::TevI in Mammalian Cells
[0313] DNA encoding the TALE-AvrBs3::TevI construct from either
pCLS9012 (SEQ ID NO: 218) or pCLS9013 (SEQ ID NO: 153) was
subcloned into the pCLS1853 (SEQ ID NO: 193) mammalian expression
plasmid using Ascl and XhoI restriction enzymes for the receiving
plasmid and BssHII and XhoI restriction enzymes for the
TALE-AvrBs3::TevI insert, leading to the mammalian expression
plasmids pCLS8993 and pCLS8994 (SEQ ID NO: 194 and 195),
respectively.
[0314] All mammalian target reporter plasmids containing the TALEN
DNA target sequences were constructed using the standard Gateway
protocol (INVITROGEN) into a CHO reporter vector (Arnould, Chames
et al. 2006, Grizot, Epinat et al. 2010). The TALE-AvrBs3::TevI
constructs were tested in an extrachromosomal assay in mammalian
cells (CHO K1) on pseudo palindromic targets in order to compare
activity with a standard TALE-AvrBs3::FokI TALEN, which requires
two binding sites for activity. AvrBs3 targets contain two
identical recognition sequences juxtaposed with the 3' ends
proximal and separated by "spacer" DNA ranging from 5 to 40 bps
(SEQ ID NO: 157 to 192, Table 6).
[0315] For this assay, CHO K1 cells were transfected in a 96-well
plate format with 75 ng of target vector and an increasing quantity
of each variant DNA from 0.7 to 25 ng, in the presence of PolyFect
reagent NIL per well). The total amount of transfected DNA was
completed to 125 ng (target DNA, variant DNA, carrier DNA) using an
empty vector. Seventy-two hours after transfection, culture medium
was removed and 150 .mu.l of lysis/revelation buffer for
.beta.-galactosidase liquid assay was added. After incubation at
37.degree. C., optical density was measured at 420 nm. The entire
process is performed on an automated Velocityll BioCel platform
(Grizot, Epinat et al. 2009).
[0316] Activity levels in mammalian cells for the TALE-AvrBs3::TevI
constructs (12.5 ng DNA transfected) on the Avr15 target (SEQ ID
NO: 167) are shown in FIG. 10. TALE-AvrBs3::TevI appears to be
efficient to cleave the target sequence.
[0317] TALE::TevI Nickase Activity
[0318] The results described in examples above illustrate two
TALE::TevI fusions, each containing one TALE-based DNA binding
domain and one I-TevI-based catalytic domain, working to generate
detectable activity. The assays used measure tandem repeat
recombination by single-strand annealing, a process that is
triggered essentially by a DSB (Sugawara and Haber 1992; Paques and
Duchateau 2007). TALE::TevI fusions can have a nickase activity
insufficient to alone trigger a signal in the cell-based assay.
However, two TALE::TevI proteins binding on two nearby sites can
sometimes generate two independent nicks, that when proximal and on
different DNA strands can create a DSB. In this case, each
TALE::TevI is a cTALEN able to generate a nick.
[0319] Different experiments are set up to measure TALE::TevI
nickase activity:
[0320] Super-Coiled Circular Plasmid Nicking and/or Linearization
Assay
[0321] The sequences encoding the TALE-AvrBs3::TevI constructs
cT11Avr_TevD01 and cT11Avr_TevD02 are cloned into a T7-based
expression vector using NcoI/EagI restriction sites to yield
plasmids pCLS9021 (SEQ ID NO: 201) and pCLS9022 (SEQ ID NO: 202),
respectively. This cloning step results in TALE-AvrBs3::TevI
proteins having an additional hexa-His tag for purification.
Plasmids pCLS9021 and pCLS9022 are then used to produce active
proteins by one of two methods: [0322] 1. Plasmids are used in a
standard in vitro transcription/translation system; lysates from
the translation are used directly without further purification.
[0323] 2. Plasmids are used to transform E. coli BL21(DE3) cells
for expression using standard protocols, namely: growth to log
phase, induction with IPTG, harvest, cell lysis and purification
via affinity methods for His-tagged proteins. Active proteins are
assayed against DNA targets having either none, one or two AvrBs3
recognition site sequences. When more than one site is present,
identical recognition sequences are juxtaposed with the 3' ends
proximal and separated by "spacer" DNA ranging from 5 to 40
bps.
[0324] A super-coiled circular plasmid nicking and/or linearization
assay is performed. Plasmids harboring the DNA targets described
above are prepared by standard methods and column purified to yield
super-coiled plasmid of >98% purity. Increasing amounts of
TALE-AvrBs3::TevI proteins (prepared as described above) are
incubated with each plasmid under conditions to promote DNA
cleavage for 1 h at 37.degree. C. Reaction products are separated
on agarose gels and visualized by EtBr staining.
[0325] Linear DNA Nicking and/or Cleavage Assay
[0326] A linear DNA nicking and/or cleavage assay is also
performed. PCR products containing the target sequences described
above are prepared by standard methods and column purified to yield
linear substrate of >98% purity. Increasing amounts of
TALE-AvrBs3::TevI proteins (prepared as described above) are then
incubated with each PCR substrate under conditions to promote DNA
cleavage for 1 h at 37.degree. C. Reaction products are separated
on a denaturing acrylamide gel and the single-strand DNA
visualized.
[0327] Engineering of the TALE::TevI
[0328] Variants differing by truncations of the C-terminal domain
of the AvrBs3-derived TALEN (SEQ ID NO: 196) are chosen as starting
scaffolds. A subset of these variants includes truncation after
positions E886 (C0), P897 (C11), G914 (C28), L926 (C40), D950
(C64), R1000 (C115), D1059 (C172) (the protein domains of truncated
C-terminal domains C11 to C172 are respectively given in SEQ ID NO:
204 to 209) and P1117 [also referred as Cter wt or WT Cter (SEQ ID
NO: 210) lacking the activation domain of the C-terminal domain of
natural AvrBs3 (SEQ ID NO: 220)]. The plasmids coding for the
variant scaffolds containing the AvrBs3-derived N-terminal domain,
the AvrBs3-derived set of repeat domains and the truncated
AvrBs3-derived C-terminal domain [pCLS7821, pCLS7803, pCLS7807,
pCLS7809, pCLS7811, pCLS7813, pCLS7817 (SEQ ID NO: 211 to 217)
which are based on the pCLS7184 (SEQ ID NO: 196)] allow cloning of
any catalytic domain in fusion to the C-terminal domain, using the
restriction sites BamHI and EagI.
[0329] Variants of the catalytic domain of I-TevI (SEQ ID NO: 20)
are designed from the N-terminal region of I-TevI. A subset of
these variants includes truncations of the catalytic domain, as the
deletion-intolerant region of its linker, the deletion-tolerant
region of its linker and its zinc finger (SEQ ID NO: 197 to 200)
named in Liu et al, 2008 (Liu, Dansereau et al. 2008).
[0330] The DNA corresponding to these variants of I-TevI is
amplified by the PCR to introduce, at the DNA level, a BamHI (at
the 5' of the coding strand) and a EagI (at the 3' of the coding
strand) restriction site and, at the protein level, a linker (for
example -SGGSGS- stretch, SEQ ID NO: 219) between the C terminal
domain of the TALE and the variant of the catalytic domain of
I-TevI. The final TALE::TevI constructs are generated by insertion
of the variant of I-TevI catalytic domains into the scaffold
variants using BamHI and EagI and standard molecular biology
procedures.
[0331] All the yeast target reporter plasmids containing the TALEN
DNA target sequences were constructed as previously described
(International PCT Applications WO 2004/067736 and in Epinat,
Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et
al. 2006; Smith, Grizot et al. 2006).
[0332] The TALE-AvrBs3::TevI constructs were tested in a yeast SSA
assay as previously described (International PCT Applications WO
2004/067736 and in Epinat, Arnould et al. 2003; Chames, Epinat et
al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al. 2006)
on pseudo palindromic targets in order to compare activity with a
standard TALE-AvrBs3::FokI TALEN (pCLS8590, SEQ ID NO: 244), which
requires two binding sites for activity. AvrBs3 targets contain two
identical recognition sequences juxtaposed with the 3' ends
proximal and separated by "spacer" DNA ranging from 5 to 40 bps
(SEQ ID NO: 157 to 192, Table 6).
Example 3b
TevI::TALE Compact TALEN
[0333] The sT2 (SEQ ID NO: 135) core TALE scaffold described in
example 3a was selected to generate pCLS7865-cTAL11_NFS1 (pCLS9008,
SEQ ID NO: 234), where NFS1 designates the amino acid sequence
-GSSG- (with underlying restriction sites BamHI and Kpn21 in the
coding DNA to facilitate cloning). Four variants of the I-TevI (SEQ
ID NO: 20) catalytic domain were amplified by the PCR on templates
TevCreD01 [SEQ ID NO: 109 protein in plasmid pCLS6614 (SEQ ID NO:
146)] using the primer pairs CMP_G001 (SEQ ID NO: 239) and CMP_G067
(SEQ ID NO: 263) or CMP_G152 (SEQ ID NO: 264), TevCreD02 [SEQ ID
NO: 110 protein in plasmid pCLS6615 (SEQ ID NO: 203)] using the
primer pair CMP_G001 (SEQ ID NO: 239) and CMP_G068 (SEQ ID NO: 240)
or TevCreD05 [SEQ ID NO: 113 protein in plasmid pCLS6618 (SEQ ID
NO: 258)] using the primer pair CMP_G001 (SEQ ID NO: 239) and
CMP_G114 (SEQ ID NO: 265) and subcloned into the pCLS9008 backbone
by restriction and ligation using NcoI and Kpn2I restriction sites,
yielding pCLS7865-TevW01_cT11 (pCLS15777, SEQ ID NO: 266, encoding
the protein of SEQ ID NO: 426), pCLS7865-TevD01_cT11 (pCLS15778,
SEQ ID NO: 267, encoding the protein of SEQ ID NO: 427),
pCLS7865-TevD02_cT11 (pCLS12730, SEQ ID NO: 235, encoding the
protein of SEQ ID NO: 428) and pCLS7865-TevD05_cT11 (pCLS15779, SEQ
ID NO: 268, encoding the protein of SEQ ID NO: 429), respectively.
Whereas the TevW01_cT11-based fusion contains the dipeptide -SG-
linking the TALE-derived DNA binding domain and I-TevI-derived
catalytic domain, all others constructs incorporate a longer
pentapeptide -QGPSG- to link the domains.
[0334] Activity of TevI::TALE in Yeast
[0335] The DNA sequence coding for the RVDs to target the AvrBs3
site (SEQ ID NO: 152) was subcloned into plasmids pCLS15777 (SEQ ID
NO: 266), pCLS15778 (SEQ ID NO: 267) and pCLS12730 (SEQ ID NO: 235)
using Type IIS restriction enzymes BsmBI for the receiving plasmid
and BbvI and SfaNI for the inserted RVD sequence to create the
subsequent TevI::TALE-AvrBs3 constructs TevW01_cT11Avr (pCLS15780,
SEQ ID NO: 269, encoding the protein of SEQ ID NO: 430),
TevD01_cT11Avr (pCLS15781, SEQ ID NO: 270, encoding the protein of
SEQ ID NO: 431) and TevD02_cT11Avr (pCLS12731, SEQ ID NO: 236,
encoding the protein of SEQ ID NO: 432), respectively. A similar
cloning technique was used to introduce the RVDs to target the
RagT2-R site (SEQ ID NO: 271) into plasmid pCLS15779 (SEQ ID NO:
268) to create the subsequent construct TevD05_cT11RagT2-R
(pCLS15782, SEQ ID NO: 272). All TevI::TALE constructs were
sequenced and the inserts transferred to additional vectors as
needed (see below).
[0336] The final TevI::TALE-based yeast expression plasmids,
pCLS11979 (SEQ ID NO: 273), pCLS8521 (SEQ ID NO: 274), pCLS8522
(SEQ ID NO: 237) and pCLS12100 (SEQ ID NO: 275), were prepared by
yeast in vivo cloning using plasmid pCLS15780 (SEQ ID NO: 269),
pCLS15781 (SEQ ID NO: 270), pCLS12731 (SEQ ID NO: 236) and
pCLS15782 (SEQ ID NO: 272), respectively. To generate an intact
coding sequence by in vivo homologous recombination, approximately
40 ng of each plasmid linearized by digestion with BssHII and 1 ng
of the pCLS0542 (SEQ ID NO: 156) plasmid DNA linearized by
digestion with NcoI and EagI were used to transform, respectively,
the yeast S. cerevisiae strain FYC2-6A (MAT.alpha., trp1.DELTA.63,
leu2.DELTA.1, his3.DELTA.200) using a high efficiency LiAc
transformation protocol (Arnould et al. 2007).
[0337] All the yeast target reporter plasmids containing the TALEN
DNA target sequences were constructed as previously described
(International PCT Applications WO 2004/067736 and in Epinat,
Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et
al. 2006; Smith, Grizot et al. 2006).
[0338] The TevI::TALE-AvrBs3 and TevI::TALE-RagT2-R constructs were
tested in a yeast SSA assay as previously described (International
PCT Applications WO 2004/067736 and in Epinat, Arnould et al. 2003;
Chames, Epinat et al. 2005; Arnould, Chames et al. 2006; Smith,
Grizot et al. 2006) on pseudo palindromic targets in order to
compare activity with a standard TALE-AvrBs3::FokI TALEN (pCLS8590,
SEQ ID NO: 244), which requires two binding sites for activity.
AvrBs3 targets contain two identical recognition sequences
juxtaposed with the 3' ends proximal and separated by "spacer" DNA
ranging from 5 to 40 bps (SEQ ID NO: 157 to 192, Table 6). In
addition, constructs were tested on a target having only a single
AvrBs3 or RagT2-R recognition site (SEQ ID NO: 238, Table 8). The
TevI::TALE-AvrBs3 activity level in yeast was comparable to that of
TALE-AvrBs3::TevI (pCLS8524, SEQ ID NO: 155) on suitable targets.
Significant activity is illustrated in table 8 for a sample
single-site target, according to the cTALEN of the present
invention.
TABLE-US-00007 TABLE 8 Activity of TevI::TALE-AvrBs3 and and
TevI::TALE- RagT2-R on dual- and single-site DNA targets. TALEN
Construct TevI:: TevI:: TALE- TALE- TALE- AvrBs3:: Target DNA
AvrBs3 RagT2-R FokI Avr25 (dual-site) ++++ n.d. ++++ (SEQ ID NO:
177) Avr25RAGT2R ++ ++ n.d. (single-site) (SEQ ID NO: 238) Relative
activity is scaled as: n.d., no activity detectable; +, <25%
activity; ++, 25% to <50% activity; +++, 50% to <75%
activity; ++++, 75% to 100% activity.
[0339] Activity of TevI::TALE in Plants
[0340] The DNA sequence coding for the RVDs to target the NptIIT5-L
and NptIIT6-L sites (SEQ ID NO: 276 to 279) were subcloned into
plasmid pCLS12730 (SEQ ID NO: 235) using Type IIS restriction
enzymes BsmBI for the receiving plasmid and BbvI and SfaNI for the
inserted RVD sequences to create the subsequent TevI::TALE
constructs TevD02_cT11NptIIT5-L (pCLS15783, SEQ ID NO: 280) and
TevD02_cT11NptIIT6-L (pCLS15784, SEQ ID NO: 281), respectively. The
constructs were sequenced and the TevI::TALE inserts transferred by
standard cloning techniques to plasmid pCLS14529 (SEQ ID NO: 282)
to generate the final TevI::TALE-NptIIT5-L and TevI::TALE-NptIIT6-L
expression plasmids, pCLS14579 (SEQ ID NO: 283) and pCLS14581 (SEQ
ID NO: 284), respectively. Plasmid pCLS14529 allows for cloning
gene of interest sequences downstream of a promoter that confers
high levels of constitutive expression in plant cells.
[0341] To test activity in plant cells, a YFP-based single-strand
annealing (SSA) assay was employed. The YFP reporter gene has a
short duplication of coding sequence that is interrupted by either
an NptIIT5 or NptIIT6 TALEN target site. Cleavage at the target
site stimulates recombination between the repeats, resulting in
reconstitution of a functional YFP gene. To quantify cleavage, the
reporter is introduced along with a construct encoding a FokI-based
TALEN or compact TALEN into tobacco protoplasts by PEG-mediated
transformation (as known or derived from the state of the art).
Uniform transformation efficiencies were obtained by using the same
amount of plasmid in each transformation--i.e. 15 .mu.g each of
plasmids encoding YFP and either the TALEN or cTALEN. After 24
hours, the protoplasts were subjected to flow cytometry to quantify
the number of YFP positive cells. The TevI::TALE activity levels,
using cTALENs according to the present invention, in plants were
comparable to those of a FokI-based TALEN control constructs on the
targets tested (Table 9).
TABLE-US-00008 TABLE 9 Activity of TevI::TALE-NptIIT5-L and
TevI::TALE- NptIIT6-L on appropriate DNA targets. TALEN Construct
TevI:: TevI:: Target TALE- NptII5.1 TALE- NptII6.1 DNA NptIIT5-L
control NptIIT6-L control NptII5.1 +++ + n.a. n.a. NptII6.1 n.a.
n.a. + + Relative activity is scaled to the control constructs as:
n.a., not applicable; +, 100% activity of control (2% YFP positive
cells).
Example 3c
TALE::NucA Compact TALEN
[0342] NucA (SEQ ID NO: 26), a nonspecific endonuclease from
Anabaena sp., was fused to a TALE-derived scaffold (composed of a
N-terminal domain, a central core composed of RVDs and a C-terminal
domain) to create a new class of cTALEN (TALE::NucA). To
distinguish the orientation (N-terminal vs. C-terminal) of the
catalytic domain (CD) fusions, construct names are written as
either CD::TALE-RVD (catalytic domain is fused N-terminal to the
TALE domain) or TALE-RVD::CD (catalytic domain is fused C-terminal
to the TALE domain), where "-RVD" optionally designates the
sequence recognized by the TALE domain and "CD" is the catalytic
domain type. Herein, we describe novel TALE::NucA constructions
that target for example the AvrBs3 sequence, and are thus named
TALE-AvrBs3::NucA. Notably, the wild-type NucA endonuclease can be
inhibited by complex formation with the NuiA protein (SEQ ID NO:
229). In a compact TALEN context, the NuiA protein can function as
an auxiliary domain to modulate the nuclease activity of TALE::NucA
constructs.
[0343] Activity of TALE::NucA in Yeast
[0344] A core TALE scaffold, sT2 (SEQ ID NO: 135), was selected
onto which (a) different sets of RVD domains could be inserted to
change DNA binding specificity, and; (b) a selection of
NucA-derived catalytic domains could be attached, N- or C-terminal,
to effect DNA cleavage (or nicking). As previously mentioned, the
sT2 truncated scaffold was generated by the PCR from a full-length
core TALEN scaffold template (pCLS7183, SEQ ID NO: 141) using
primers CMP_G061 (SEQ ID NO: 142) and CMP_G065 (SEQ ID NO: 143) and
was cloned into vector pCLS7865 (SEQ ID NO: 144) to generate
pCLS7865-cTAL11_CFS1 (pCLS9009, SEQ ID NO: 145), where CFS1
designates the amino acid sequence -GSSG- (with underlying
restriction sites BamHI and Kpn2I in the coding DNA to facilitate
cloning). The NucA (SEQ ID NO: 26) catalytic domain, corresponding
to amino acid residues 25 to 274, was subcloned into the pCLS9009
backbone (SEQ ID NO: 145) by restriction and ligation using BamHI
and EagI restriction sites, yielding pCLS7865-cT11_NucA (pCLS9937,
SEQ ID NO: 221, encoding the protein of SEQ ID NO: 433). The fusion
contains the dipeptide -GS- linking the TALE-derived DNA binding
domain and NucA-derived catalytic domain. The cloning step also
brings at the amino acid level an AAD sequence at the Cter of the
NucA catalytic domain.
[0345] The DNA sequence coding for the RVDs to target the AvrBs3
site (SEQ ID NO: 152) was subcloned into plasmid pCLS9937 (SEQ ID
NO: 221) using Type IIS restriction enzymes BsmBI for the receiving
plasmid and BbvI and SfaNI for the inserted RVD sequence to create
the subsequent TALE-AvrBs3::NucA construct cT11Avr_NucA (pCLS9938,
SEQ ID NO: 222, encoding the protein of SEQ ID NO: 434). The
TALE-AvrBs3::NucA construct was sequenced and the insert
transferred to additional vectors as needed (see below).
[0346] The final TALE-AvrBs3::NucA yeast expression plasmid,
pCLS9924 (SEQ ID NO: 223), was prepared by yeast in vivo cloning
using plasmid pCLS9938 (SEQ ID NO: 222). To generate an intact
coding sequence by in vivo homologous recombination, approximately
40 ng of plasmid (pCLS9938) linearized by digestion with BssHII and
1 ng of the pCLS0542 (SEQ ID NO: 156) plasmid DNA linearized by
digestion with NcoI and EagI were used to transform the yeast S.
cerevisiae strain FYC2-6A (MAT.alpha., trp1.DELTA.63, leu2.DELTA.1,
his3.DELTA.200) using a high efficiency LiAc transformation
protocol (Arnould et al. 2007).
[0347] All the yeast target reporter plasmids containing the TALEN
DNA target sequences were constructed as previously described
(International PCT Applications WO 2004/067736 and in Epinat,
Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et
al. 2006; Smith, Grizot et al. 2006).
[0348] The TALE-AvrBs3::NucA construct was tested in a yeast SSA
assay as previously described (International PCT Applications WO
2004/067736 and in Epinat, Arnould et al. 2003; Chames, Epinat et
al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al. 2006)
on pseudo palindromic targets in order to compare activity with a
standard TALE-AvrBs3::FokI TALEN (pCLS8590, SEQ ID NO: 244), which
requires two binding sites for activity. AvrBs3 targets contain two
identical recognition sequences juxtaposed with the 3' ends
proximal and separated by "spacer" DNA ranging from 5 to 40 bps
(SEQ ID NO: 157 to 192, Table 7). In addition, constructs were
tested on a target having only a single AvrBs3 recognition site
(SEQ ID NO: 224; Table 7).
[0349] Engineering of the TALE::NucA
[0350] Variants differing by truncations of the C-terminal domain
of the AvrBs3-derived TALEN (SEQ ID NO: 196) are chosen as starting
scaffolds. A subset of these variants includes truncation after
positions E886 (C0), P897 (C11), G914 (C28), L926 (C40), D950
(C64), R1000 (C115), D1059 (C172) (the protein domains of truncated
C-terminal domains C11 to C172 are respectively given in SEQ ID NO:
204 to 209) and P1117 [also referred as Cter wt or WT Cter (SEQ ID
NO: 210) lacking the activation domain of the C-terminal domain of
natural AvrBs3 (SEQ ID NO: 220)]. The plasmids coding for the
variant scaffolds containing the AvrBs3-derived N-terminal domain,
the AvrBs3-derived set of repeat domains and the truncated
AvrBs3-derived C-terminal domain [pCLS7821, pCLS7803, pCLS7807,
pCLS7809, pCLS7811, pCLS7813, pCLS7817 (SEQ ID NO: 211 to 217)
which are based on the pCLS7184 (SEQ ID NO: 196)] allow cloning of
any catalytic domain in fusion to the C-terminal domain, using the
restriction sites BamHI and EagI.
[0351] The DNA corresponding to amino acid residues 25 to 274 of
NucA is amplified by the PCR to introduce, at the DNA level, a
BamHI (at the 5' of the coding strand) and a EagI (at the 3' of the
coding strand) restriction site and, at the protein level, a linker
(for example -SGGSGS- stretch, SEQ ID NO: 219) between the C
terminal domain of the TALE and the NucA catalytic domain. The
final TALE::NucA constructs are generated by insertion of the NucA
catalytic domain into the scaffold variants using BamHI and EagI
and standard molecular biology procedures. For example, scaffold
variants truncated after positions P897 (C11), G914 (C28) and D950
(C64), respectively encoded by pCLS7803, pCLS7807, pCLS7811, (SEQ
ID NO: 212, 213 and 215), were fused to the NucA catalytic domain
(SEQ ID NO: 26), leading to pCLS9596, pCLS9597, and pCLS9599 (SEQ
ID NO: 225 to 227). The cloning step also brings at the amino acid
level an AAD sequence at the Cter of the NucA catalytic domain.
[0352] All the yeast target reporter plasmids containing the TALEN
DNA target sequences were constructed as previously described
(International PCT Applications WO 2004/067736 and in Epinat,
Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et
al. 2006; Smith, Grizot et al. 2006).
[0353] The TALE-AvrBs3::NucA constructs were tested in a yeast SSA
assay as previously described (International PCT Applications WO
2004/067736 and in Epinat, Arnould et al. 2003; Chames, Epinat et
al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al. 2006)
on pseudo palindromic targets in order to compare activity with a
standard TALE-AvrBs3::FokI TALEN (pCLS8590, SEQ ID NO: 244), which
requires two binding sites for activity. AvrBs3 targets contain two
identical recognition sequences juxtaposed with the 3' ends
proximal and separated by "spacer" DNA ranging from 5 to 40 bps
(SEQ ID NO: 157 to 192, Table 7). In addition, TALE-AvrBs3::NucA
constructs were tested on a target having only a single AvrBs3
recognition site (SEQ ID NO: 224). Data summarized in FIG. 11 show
that TALE-AvrBs3::NucA constructs are active on all targets having
at least one AvrBs3 recognition site, according to the cTALEN of
the present invention.
Example 3d
TALE::ColE7 Compact TALEN
[0354] The catalytic domain of ColE7 (SEQ ID NO: 140), a
nonspecific endonuclease from E. coli, was fused to a TALE-derived
scaffold (composed of a N-terminal domain, a central core composed
of RVDs and a C-terminal domain) to create a new class of cTALEN
(TALE::ColE7). To distinguish the orientation (N-terminal vs.
C-terminal) of the catalytic domain (CD) fusions, construct names
are written as either CD::TALE-RVD (catalytic domain is fused
N-terminal to the TALE domain) or TALE-RVD::CD (catalytic domain is
fused C-terminal to the TALE domain), where "-RVD" optionally
designates the sequence recognized by the TALE domain and "CD" is
the catalytic domain type. Herein, we describe novel TALE::ColE7
constructions that target for example the AvrBs3 sequence, and are
thus named TALE-AvrBs3::ColE7. Notably, the wild-type ColE7
endonuclease can be inhibited by complex formation with the Im7
immunity protein (SEQ ID NO: 230). In a compact TALEN context, the
Im7 protein can function as an auxiliary domain to modulate the
nuclease activity of TALE::ColE7 constructs.
[0355] Activity of TALE::ColE7 in Yeast
[0356] A core TALE scaffold, sT2 (SEQ ID NO: 135), was selected
onto which (a) different sets of RVD domains could be inserted to
change DNA binding specificity, and; (b) a selection of
ColE7-derived catalytic domains could be attached, N- or
C-terminal, to effect DNA cleavage (or nicking). As previously
mentioned, the sT2 truncated scaffold was generated by the PCR from
a full-length core TALEN scaffold template (pCLS7183, SEQ ID NO:
141) using primers CMP_G061 (SEQ ID NO: 142) and CMP_G065 (SEQ ID
NO: 143) and was cloned into vector pCLS7865 (SEQ ID NO: 144) to
generate pCLS7865-cTAL11_CFS1 (pCLS9009, SEQ ID NO: 145), where
CFS1 designates the amino acid sequence -GSSG- (with underlying
restriction sites BamHI and Kpn21 in the coding DNA to facilitate
cloning). The ColE7 (SEQ ID NO: 140) catalytic domain was subcloned
into the pCLS9009 backbone by restriction and ligation using Kpn2I
and EagI restriction sites, yielding pCLS7865-cT11_ColE7 (pCLS9939,
SEQ ID NO: 231, encoding the protein of SEQ ID NO: 435). The fusion
contains the dipeptide -GSSG- linking the TALE-derived DNA binding
domain and ColE7-derived catalytic domain.
[0357] The DNA sequence coding for the RVDs to target the AvrBs3
site (SEQ ID NO: 152) was subcloned into plasmid pCLS9939 (SEQ ID
NO: 231) using Type IIS restriction enzymes BsmBI for the receiving
plasmid and BbvI and SfaNI for the inserted RVD sequence to create
the subsequent TALE-AvrBs3::ColE7 construct cT11Avr_ColE7
(pCLS9940, SEQ ID NO: 232, encoding the protein of SEQ ID NO: 436).
The TALE-AvrBs3::ColE7 construct was sequenced and the insert
transferred to additional vectors as needed (see below).
[0358] The final TALE-AvrBs3::ColE7 yeast expression plasmid,
pCLS8589 (SEQ ID NO: 233), was prepared by yeast in vivo cloning
using plasmid pCLS9940 (SEQ ID NO: 232). To generate an intact
coding sequence by in vivo homologous recombination, approximately
40 ng of plasmid (pCLS9940) linearized by digestion with BssHII and
1 ng of the pCLS0542 (SEQ ID NO: 156) plasmid DNA linearized by
digestion with NcoI and EagI were used to transform the yeast S.
cerevisiae strain FYC2-6A (MAT.alpha., trp1.DELTA.63, leu2.DELTA.1,
his3.DELTA.200) using a high efficiency LiAc transformation
protocol (Arnould et al. 2007).
[0359] All the yeast target reporter plasmids containing the TALEN
DNA target sequences were constructed as previously described
(International PCT Applications WO 2004/067736 and in Epinat,
Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et
al. 2006; Smith, Grizot et al. 2006).
[0360] The TALE-AvrBs3::ColE7 construct was tested in a yeast SSA
assay as previously described (International PCT Applications WO
2004/067736 and in Epinat, Arnould et al. 2003; Chames, Epinat et
al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al. 2006)
on pseudo palindromic targets in order to compare activity with a
standard TALE-AvrBs3::FokI TALEN (pCLS8590, SEQ ID NO: 244), which
requires two binding sites for activity. AvrBs3 targets contain two
identical recognition sequences juxtaposed with the 3' ends
proximal and separated by "spacer" DNA ranging from 5 to 40 bps
(SEQ ID NO: 157 to 192, Table 7). In addition, constructs were
tested on a target having only a single AvrBs3 recognition site
(SEQ ID NO: 224, Table 7). TALE-AvrBs3::ColE7 activity levels on
the respective targets in yeast cells are shown in FIG. 12.
[0361] Activity of TALE::ColE7 in Plants
[0362] The DNA sequence coding for the RVDs to target the NptIIT5-L
and NptIIT6-L sites (SEQ ID NO: 276 to 279) were subcloned into
plasmid pCLS15785 (SEQ ID NO: 285, a C-terminally modified ColE7
K497A mutant of plasmid pCLS9939, SEQ ID NO: 231) using Type IIS
restriction enzymes BsmBI for the receiving plasmid and BbvI and
SfaNI for the inserted RVD sequences to create the subsequent
TALE::ColE7_A497 constructs cT11NptIIT5-L_ColE7_A497 (pCLS15786,
SEQ ID NO: 286) and cT11NptIIT6-L_ColE7_A497 (pCLS15787, SEQ ID NO:
287), respectively. The constructs were sequenced and the
TALE::ColE7_A497 inserts transferred by standard cloning techniques
to plasmid pCLS14529 (SEQ ID NO: 282) to generate the final
TALE-NptIIT5-L::ColE7_A497 and TALE-NptIIT6-L::ColE7_A497
expression plasmids, pCLS14584 (SEQ ID NO: 288, encoding the
protein of SEQ ID NO: 437) and pCLS14587 (SEQ ID NO: 289, encoding
the protein of SEQ ID NO: 438), respectively. Plasmid pCLS14529
allows for cloning gene of interest sequences downstream of a
promoter that confers high levels of constitutive expression in
plant cells.
[0363] To test activity in plant cells, a YFP-based single-strand
annealing (SSA) assay was employed. The YFP reporter gene has a
short duplication of coding sequence that is interrupted by either
an NptIIT5 or NptIIT6 TALEN target site. Cleavage at the target
site stimulates recombination between the repeats, resulting in
reconstitution of a functional YFP gene. To quantify cleavage, the
reporter is introduced along with a construct encoding a FokI-based
TALEN or compact TALEN into tobacco protoplasts by PEG-mediated
transformation. Uniform transformation efficiencies were obtained
by using the same amount of plasmid in each transformation--i.e. 15
.mu.g each of plasmids encoding YFP and either the TALEN or cTALEN.
After 24 hours, the protoplasts were subjected to flow cytometry to
quantify the number of YFP positive cells. The TALE:: ColE7_A497
activity levels, using cTALENs according to the present invention,
in plants were comparable to those of a FokI-based TALEN control
constructs on the targets tested (Table 10).
TABLE-US-00009 TABLE 10 Activity of TALE-NptIIT5-L::ColE7_A497 and
TALE-NptIIT6- L::ColE7_A497 on appropriate DNA targets. TALEN
Construct TALE- TALE- Target NptIIT5-L:: NptII5.1 NptIIT6-L::
NptII6.1 DNA ColE7_A497 control ColE7_A497 control NptII5.1 + +
n.a. n.a. NptII6.1 n.a. n.a. + + Relative activity is scaled to the
control constructs as: n.a., not applicable; +, 100% activity of
control (8% YFP positive cells).
[0364] Engineering of the TALE::ColE7
[0365] Variants differing by truncations of the C-terminal domain
of the AvrBs3-derived TALEN (SEQ ID NO: 196) are chosen as starting
scaffolds. A subset of these variants includes truncation after
positions E886 (C0), P897 (C11), G914 (C28), L926 (C40), D950
(C64), R1000 (C115), D1059 (C172) (the protein domains of truncated
C-terminal domains C11 to C172 are respectively given in SEQ ID NO:
204 to 209) and P1117 [also referred as Cter wt or WT Cter (SEQ ID
NO: 210) lacking the activation domain of the C-terminal domain of
natural AvrBs3 (SEQ ID NO: 220)]. The plasmids coding for the
variant scaffolds containing the AvrBs3-derived N-terminal domain,
the AvrBs3-derived set of repeat domains and the truncated
AvrBs3-derived C-terminal domain [pCLS7821, pCLS7803, pCLS7807,
pCLS7809, pCLS7811, pCLS7813, pCLS7817 (SEQ ID NO: 211 to 217)
which are based on the pCLS7184 (SEQ ID NO: 196)] allow cloning of
any catalytic domain in fusion to the C-terminal domain, using the
restriction sites BamHI and EagI.
[0366] The DNA corresponding to the catalytic domain of ColE7 is
amplified by the PCR to introduce, at the DNA level, a BamHI (at
the 5' of the coding strand) and a EagI (at the 3' of the coding
strand) restriction site and, at the protein level, a linker (for
example -SGGSGS- stretch, SEQ ID NO: 219) between the C terminal
domain of the TALE and the ColE7 catalytic domain. Additionally,
variants of the ColE7 endonuclease domain that modulate catalytic
activity can be generated having changes (individually or combined)
at the following positions: K446, R447, D493, R496, K497, H545,
N560 and H573 [positions refer to the amino acid sequence of the
entire ColE7 protein (SEQ ID NO: 11)]. The final TALE::ColE7
constructs are generated by insertion of the ColE7 catalytic domain
into the scaffold variants using BamHIH and EagI and standard
molecular biology procedures.
[0367] All the yeast target reporter plasmids containing the TALEN
DNA target sequences were constructed as previously described
(International PCT Applications WO 2004/067736 and in Epinat,
Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et
al. 2006; Smith, Grizot et al. 2006).
[0368] The TALE-AvrBs3::ColE7 constructs are tested in a yeast SSA
assay as previously described (International PCT Applications WO
2004/067736 and in Epinat, Arnould et al. 2003; Chames, Epinat et
al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al. 2006)
on pseudo palindromic targets in order to compare activity with a
standard TALE-AvrBs3::FokI TALEN (pCLS8590, SEQ ID NO: 244), which
requires two binding sites for activity. AvrBs3 targets contain two
identical recognition sequences juxtaposed with the 3' ends
proximal and separated by "spacer" DNA ranging from 5 to 40 bps
(SEQ ID NO: 157 to 192, Table 7). In addition, constructs were
tested on a target having only a single AvrBs3 recognition site
(SEQ ID NO: 224, Table 7).
Example 3e
TALE::CreI Compact TALEN
[0369] The wild-type I-CreI meganuclease (SEQ ID NO: 106) was
chosen as a protein template to derive a sequence-specific
catalytic domain that when fused to a TALE-derived scaffold
(composed of a N-terminal domain, a central core composed of RVDs
and a C-terminal domain) would generate a new class of cTALEN
(TALE::CreI). To distinguish the orientation (N-terminal vs.
C-terminal) of the catalytic domain (CD) fusions, construct names
are written as either CD::TALE-RVD (catalytic domain is fused
N-terminal to the TALE domain) or TALE-RVD::CD (catalytic domain is
fused C-terminal to the TALE domain), where "-RVD" optionally
designates the sequence recognized by the TALE domain and "CD" is
the catalytic domain type. Herein, we describe novel
TALE::CreI-based constructions that target for example the T cell
receptor B gene (TCRB gene, SEQ ID NO: 290, FIG. 13) sequence, both
via the TALE DNA binding domain and the re-engineered I-CreI
domain. Notably, specificity of the TALE::CreI compact TALEN is
driven by both the TALE DNA binding domain as well as the
I-CreI-derived catalytic domain. In a compact TALEN context, such
proteins can provide, within a reasonably-sized monomeric protein,
the requisite high specificity demanded by therapeutic
applications.
[0370] Activity of TALE::CreI in Yeast
[0371] A core TALE scaffold, sT2 (SEQ ID NO: 135), was selected
onto which (a) different sets of RVD domains could be inserted to
change DNA binding specificity, and; (b) a selection of
I-CreI-derived catalytic domains could be attached, N- or
C-terminal, to effect DNA cleavage (or nicking). As previously
mentioned, the sT2 truncated scaffold was generated by the PCR from
a full-length core TALEN scaffold template (pCLS7183, SEQ ID NO:
141) using primers CMP_G061 (SEQ ID NO: 142) and CMP_G065 (SEQ ID
NO: 143) and was cloned into vector pCLS7865 (SEQ ID NO: 144) to
generate pCLS7865-cTAL11_CFS1 (pCLS9009, SEQ ID NO: 145), where
CFS1 designates the amino acid sequence -GSSG- (with underlying
restriction sites BamHI and Kpn21 in the coding DNA to facilitate
cloning). A re-engineered I-CreI catalytic domain, designed to
target a sequence in the T cell receptor B gene (TCRB gene, SEQ ID
NO: 290, FIG. 13), was subcloned in two steps. First, the
I-CreI_NFS1 (SEQ ID NO: 122) scaffold, where NFS1 (SEQ ID NO: 98)
comprises a linker of 20 amino acids -GSDITKSKISEKMKGQGPSG- (with
underlying restriction sites BamHI and Kpn21 in the coding DNA to
facilitate cloning), was fused to the pCLS7865-cTAL11_CFS1 scaffold
(using BamHIH and EagI restriction sites) to insert the NFS1 linker
in-frame to the coding sequence. The I-CreI meganuclease was
subsequently replaced by the engineered TCRB02-A meganuclease
(pCLS6857, SEQ ID NO: 291) construct using Kpn2I and XhoI
restriction sites, yielding pCLS7865-cT11_scTB2aD01 (pCLS15788, SEQ
ID NO: 292, encoding the protein of SEQ ID NO: 439). Two
point-mutant variants of the TCRB02-A meganuclease,
TCRB02-A.sub.--148C (pCLS12083, SEQ ID NO: 293, encoding the
protein of SEQ ID NO: 442) and TCRB02-A.sub.--333C (pCLS12195, SEQ
ID NO: 294, encoding the protein of SEQ ID NO: 443), were also
subcloned as catalytic domains fused to a TALE binding core,
yielding constructs pCLS7865-cT11_scTB2aD01.sub.--148C (pCLS15789,
SEQ ID NO: 295, encoding the protein of SEQ ID NO: 440) and
pCLS7865-cT11_scTB2aD01.sub.--333C (pCLS15790, SEQ ID NO: 296,
encoding the protein of SEQ ID NO: 441).
[0372] Three DNA sequences coding for RVDs that target the TCRB
gene were designed at different distances from the meganuclease
site, leading to RVDs TCRBO2A1 (SEQ ID NO: 297), TCRB02A2 (SEQ ID
NO: 298) and TCRBO2A3 (SEQ ID NO: 299) that target sequences
located 7 bp, 12 by and 16 bp, respectively, upstream of the
meganuclease TCRB site (FIG. 13). DNA sequences for each RVD were
independently subcloned into plasmid pCLS15788 (SEQ ID NO: 292)
using Type IIS restriction enzymes BsmBI for the receiving plasmid
and BbvI and SfaNI for the inserted RVD sequence to create the
subsequent TALE::scTB2aD01 constructs cT11TB2A1_scTB2aD01
(pCLS15791, SEQ ID NO: 300), cT11TB2A2_scTB2aD01 (pCLS15792, SEQ ID
NO: 301) and cT11TB2A3_scTB2aD01 (pCLS15793, SEQ ID NO: 302).
Additionally, the TCRBO2A2 (SEQ ID NO: 298) RVDs were similarly
cloned into pCLS15789 (SEQ ID NO: 295) to create
cT11TB2A2_scTB2aD01.sub.--148C (pCLS15794, SEQ ID NO: 303). All
constructs were sequenced and the various inserts transferred to
additional vectors as needed (see below).
[0373] The final TALE::scTB2aD01 yeast expression plasmids,
pCLS13449 (SEQ ID NO: 304, encoding the protein of SEQ ID NO: 444),
pCLS13450 (SEQ ID NO: 305, encoding the protein of SEQ ID NO: 445),
pCLS13451 (SEQ ID NO: 306, encoding the protein of SEQ ID NO: 446)
and pCLS15148 (SEQ ID NO: 307, encoding the protein of SEQ ID NO:
455), were prepared by yeast in vivo cloning using plasmids
pCLS15791 (SEQ ID NO: 300), pCLS15792 (SEQ ID NO: 301), pCLS15793
(SEQ ID NO: 302) and pCLS15794 (SEQ ID NO: 303), respectively. To
generate an intact coding sequence by in vivo homologous
recombination, approximately 40 ng of each plasmid linearized by
digestion with BssHII and 1 ng of the pCLS0542 (SEQ ID NO: 156)
plasmid DNA linearized by digestion with NcoI and EagI were used to
transform, respectively, the yeast S. cerevisiae strain FYC2-6A
(MAT.alpha., trp1.DELTA.63, leu2.DELTA.1, his3.DELTA.200) using a
high efficiency LiAc transformation protocol (Arnould et al.
2007).
[0374] All the yeast target reporter plasmids containing the TALEN
or meganuclease DNA target sequences were constructed as previously
described (International PCT Applications WO 2004/067736 and in
Epinat, Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould,
Chames et al. 2006; Smith, Grizot et al. 2006).
[0375] The TALE::scTB2aD01-based constructs were tested in a yeast
SSA assay as previously described (International PCT Applications
WO 2004/067736 and in Epinat, Arnould et al. 2003; Chames, Epinat
et al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al.
2006) on hybrid targets TCRBO2Tsp7 (SEQ ID NO: AC4), TCRBO2Tsp12
(SEQ ID NO: AC5) and TCRBO2Tsp16 (SEQ ID NO: AC6), illustrated in
FIG. 14. The TCRB02.1-only target was included to compare activity
with the engineered TCRB02-A meganuclease (pCLS6857, SEQ ID NO:
291), which does not require the TALE DNA binding sites for
activity. Activity levels on the respective targets in yeast cells
for the indicated TALE::scTB2aD01-based constructs are shown in
FIG. 14. Notably, under the in vivo conditions tested the
TALE-TB2A2::scTB2aD01.sub.--148C (pCLS15794, SEQ ID NO: 303)
construct no longer cleaves targets lacking the DNA sequence
recognized by the TALE DNA binding moiety.
[0376] Activity of TALE::CreI in Mammalian Cells
[0377] DNA encoding the TALE-TB2A2::scTB2aD01 and
TALE-TB2A3::scTB2aD01 constructs from pCLS15792 (SEQ ID NO: 301)
and pCLS15793 (SEQ ID NO: 302) were subcloned into the pCLS1853
(SEQ ID NO: 193) mammalian expression plasmid using Ascl and XhoI
restriction enzymes for the receiving plasmid and BssHII and XhoI
restriction enzymes for TALE::scTB2aD01-based inserts, leading to
the mammalian expression plasmids pCLS14894 and pCLS14895 (SEQ ID
NO: 308 and 309), respectively.
[0378] All mammalian target reporter plasmids containing the TALEN
DNA target sequences were constructed using the standard Gateway
protocol (INVITROGEN) into a CHO reporter vector (Arnould, Chames
et al. 2006, Grizot, Epinat et al. 2010).
[0379] To monitor protein expression levels, TALE::scTB2aD01-based
constructs were transfected in mammalian cells (HEK293) alongside
the engineered TCRB02-A meganuclease (pCLS6857, SEQ ID NO: 291).
Briefly, cells were transfected, respectively, with 300 ng of each
protein encoding plasmid in the presence of lipofectamine.
Fourty-eight hours post-transfection, 20 .mu.g of total protein
extract for each sample was analyzed by Western-Blot using a
polyclonal anti-1-CreI antibody. A typical western-blot is shown in
FIG. 15.
[0380] Relative toxicity of the TALE::scTB2aD01-based constructs
was assessed using a cell survival assay. CHOK1 cells were used to
seed plates at a density of 2.5*10.sup.3 cells per well. The
following day, varying amounts of plasmid encoding either the
TALE::scTB2aD01-based constructs (pCLS14894 and pCLS14895; SEQ ID
NO: 308 and 309) or the engineered TCRB02-A meganuclease (pCLS6857,
SEQ ID NO: 291) and a constant amount of GFP-encoding plasmid (10
ng) were used to transfect the cells with a total quantity of 200
ng using Polyfect reagent. GFP levels were monitored by flow
cytometry (Guava Easycyte, Guava technologies) on days 1 and 6
post-transfection. Cell survival is expressed as a percentage,
calculated as a ratio (TALEN and meganuclease-transfected cells
expressing GFP on Day 6/control-transfected cells expressing GFP on
Day 6) corrected for the transfection efficiency determined on Day
1. Typical cell survival assay data are shown in FIG. 16.
[0381] Cleavage activity in vivo was monitored via detection of
NHEJ events in the presence of TREX2 exonuclease. Plasmid (3 .mu.g)
encoding either the TALE::scTB2aD01-based constructs (pCLS14894 and
pCLS14895; SEQ ID NO: 308 and 309) or the engineered TCRB02-A
meganuclease (pCLS6857, SEQ ID NO: 291) and 2 .mu.g of
scTrex2-encoding plasmid (pCLS8982, SEQ ID NO: 310) were used to
transfect the HEK293 cells in the presence of lipofectamine.
Genomic DNA was extracted 2 and 7 days post-transfection with the
DNeasy Blood and Tissue kit (Qiagen) and the region encompassing
the TCRB02 site (FIG. 13) was amplified using the PCR with oligos
TRBC2F3 (Seq ID NO: 311) and TRBC2R3B (SEQ ID NO: 312) at day 2
post-transfection and with oligos TRBC2F4 (SEQ ID NO: 315) and
TRBC2R4B (SEQ ID NO: 314) at day 7 post-transfection. Respective
PCR products (100 ng) were heat denatured, allowed to re-anneal by
slow-cooling then treated with T7 endonuclease 1 (NEB) for 15
minutes at 37.degree. C. Digested PCR products are separated on 10%
acrylamide gels and visualized with SYBRgreen (Invitrogen)
staining. Cleavage of mismatched DNA sequences by T7 endonuclease
is indicative of NHEJ events resulting from the activity of the
cTALEN or meganuclease at the targeted locus. FIG. 17 illustrates
the detectable NHEJ activity of the TALE::scTB2aD01-based
constructs (pCLS14894 and pCLS14895; SEQ ID NO: 308 and 309)
compared to the engineered TCRB02-A meganuclease (pCLS6857, SEQ ID
NO: 291). Whereas at day 2 NHEJ results are comparable for all
constructs, NHEJ activity at day 7 can only be detected for the
TALE::scTB2aD01-based constructs, suggesting that these compact
TALENs do not induce cytotoxicity.
[0382] Engineering of the TALE::CreI
[0383] A significant novel property of the TALE::CreI compact TALEN
resides in the ability to independently engineer the "hybrid"
specificity of the final molecule. As such, the inherent
activity/specificity ratio can be modulated within the
TALE::CreI-derived constructs, allowing for unprecedented specific
targeting with retention of high DNA cleavage activity. In its
simplest form, successful re-targeting of the TALE DNA binding
domain is achieved via the RVD cipher (FIG. 3), with a pseudo
one-to-one correspondence to the underlying DNA base. Engineering
of the I-CreI moiety, however, presents more challenges insomuch as
there exists a potential codependence of protein-DNA contacts
needed for suitable DNA binding and cleavage activity. Methods have
been described (WO2006097854, WO2008093249, WO03078619,
WO2009095793, WO 2007/049095, WO 2007/057781, WO 2006/097784, WO
2006/097853, WO 2007/060495, WO 2007/049156 and WO 2004/067736) to
successfully re-engineer the I-CreI meganuclease to target novel
DNA sequences. As some of these methods rely on a clustered
approach, it can be envisioned that using said approach the
"absolute" specificity of the I-CreI moiety could be reduced in a
stepwise manner. For example, the breakdown of the I-CreI DNA
interaction surface into discrete 10NNN, 7NN, 5NNN and 2NN regions
(per monomeric subunit half) allows for novel engineering wherein
high specificity is maintained in the central 5NNN-2NN region at
the expense of "loose" or broad specificity in the outer 10NNN-7NN
regions. In essence such an approach could reduce the complexity of
re-engineering the I-CreI-derived scaffold for a compact TALEN
context as only "selectivity" in cleavage is required for the
catalytic domain, with subsequent specificity provided by the TALE
DNA binding part of the protein fusion. Taken together, the ease of
engineering combined with the potential high specificity and high
DNA cleavage activity make TALE::CreI-derived compact TALENs ideal
tools for therapeutic applications. Finally, it should be noted
that the I-CreI moiety could in principle be replaced with a host
of naturally existing or re-engineered homing endonuclease-derived
catalytic domains.
Example 3f
Activity of TALE::SnaseSTAUU
[0384] Activity of TALE::SnaseSTAUU in Yeast
[0385] Variants differing by truncations of the C-terminal domain
of the AvrBs3-derived TALEN (SEQ ID Na: 196) are chosen as starting
scaffolds. A subset of these variants includes truncation after
positions G914 (C28) and L926 (C40) (the protein domains of
truncated C-terminal domains C28 and C40 are respectively given in
SEQ ID NO: 205 and 206). The plasmids coding for the variant
scaffolds containing the AvrBs3-derived N-terminal domain, the
AvrBs3-derived set of repeat domains and the truncated
AvrBs3-derived C-terminal domain [pCLS7807 and pCLS7809, (SEQ ID
NO: 213 and 214) which are based on the pCLS7184 (SEQ ID Na: 196)]
allow cloning of any catalytic domain in fusion to the C-terminal
domain, using the restriction sites BamHI and EagI.
[0386] The DNA corresponding to amino acid residues 83 to 231 of
SnaseSTAAU (SEQ ID NO: 30) is amplified by the PCR to introduce, at
the DNA level, a BamHI (at the 5' of the coding strand) and a EagI
(at the 3' of the coding strand) restriction site and, at the
protein level, a linker (for example -SGGSGS- stretch, SEQ ID NO:
219) between the C terminal domain of the TALE and the SnaseSTAAU
catalytic domain. The final TALE::SnaseSTAAU constructs are
generated by insertion of the SnaseSTAAU catalytic domain into the
scaffold variants using BamHI and EagI and standard molecular
biology procedures. Scaffold variants truncated after positions
G914 (C28) and L926 (C40), respectively encoded by pCLS7807 and
pCLS7809, (SEQ ID NO: 213 and 214), were fused to the SnaseSTAAU
catalytic domain (SEQ ID NO: 30), leading to pCLS9082 and pCLS9081
(SEQ ID NO: 370 and 371). The cloning step also brings at the amino
acid level an AAD sequence at the Cter of the SnaseSTAAU catalytic
domain.
[0387] All the yeast target reporter plasmids containing the TALEN
DNA target sequences were constructed as previously described
(International PCT Applications WO 2004/067736 and in Epinat,
Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et
al. 2006; Smith, Grizot et al. 2006).
[0388] The TALE-AvrBs3:: SnaseSTAAU constructs were tested in a
yeast SSA assay as previously described (International PCT
Applications WO 2004/067736 and in Epinat, Arnould et al. 2003;
Chames, Epinat et al. 2005; Arnould, Chames et al. 2006; Smith,
Grizot et al. 2006) on pseudo palindromic targets in order to
compare activity with a standard TALE-AvrBs3::FokI TALEN, which
requires two binding sites for activity. AvrBs3 targets contain two
identical recognition sequences juxtaposed with the 3' ends
proximal and separated by "spacer" DNA ranging from 5 to 40 bps
(SEQ ID NO: 157 to 192, Table 7). In addition,
TALE-AvrBs3::SnaseSTAAU constructs were tested on a target having
only a single AvrBs3 recognition site (SEQ ID NO: 224). Data
summarized in FIG. 19 show that TALE-AvrBs3::SnaseSTAAU constructs
are active on targets having two AvrBs3 recognition site, according
to the chimeric protein of the present invention, but also on
targets containing only one AvrBs3 recognition site.
Example 4
[0389] Basic cTALENs are composed of a single DNA binding domain
fused to a single catalytic domain and are designed to stimulate HR
via a single double-strand DNA cleavage or single-strand nicking
event. For certain applications (e.g. gene inactivation), it is
favorable to enhance the level of NHEJ. This example illustrates
the creation of a dual-cleavage cTALEN (dcTALEN) that is capable of
effecting cleavage of double-strand DNA at two distinct sites
flanking the TALE DNA binding domain (FIG. 5C). The simultaneous
cleavage of the DNA at the two sites is expected to eliminate the
intervening sequence and therefore abolish "scarless" re-ligation
by NHEJ (FIG. 1).
[0390] The baseline scaffolds (SEQ ID NO: 136 to SEQ ID NO: 139)
described in Example 3 are used as starting points for fusion
designs. A non-exhaustive list of catalytic domains amenable to
fusion with TALE DNA binding domains is presented in Table 2. A
non-exhaustive list of linkers that can be used is presented in
Table 3. See examples 3, 5, 6 and 7 for additional details
concerning the choice of linker or enhancement domain. For the
dcTALEN designs, at least one cleavase domain is fused (N- or
C-terminal) to the TALE DNA binding domain. The additional
catalytic domain can be either a nickase of cleavase (endonuclease
or exonuclease) domain, and depends on the nature of the
application. For example, the coupling of a cleavase domain on one
side with a nickase domain on the other could result in excision of
a single-strand of DNA spanning the TALE DNA binding region. The
targeted generation of extended single-strand overhangs could be
applied in applications that target DNA repair mechanisms. For
targeted gene inactivation, the use of two cleavase domains in the
dcTALEN is preferred.
[0391] All dcTALEN designs are assessed using our yeast assay (see
Example 1) and provide detectable activity comparable to existing
engineered meganucleases. Furthermore, potential enhancements in
NHEJ are monitored using the mammalian cell based assay as
described in Example 3.
Example 4a
Activity of TevI::TALE::FokI and TevI::TALE::TevI Dual Cleavage
TALENs
[0392] Dual cleavage TALENs (CD::TALE::CD), possessing an
N-terminal I-TevI-derived catalytic domain and a C-terminal
catalytic domain derived from either FokI (SEQ ID NO:368) or I-TevI
(SEQ ID NO: 20), were generated on the baseline bT2-Avr (SEQ ID NO:
137) scaffold. The catalytic domain fragment of I-TevI was excised
from plasmid pCLS12731 (SEQ ID NO: 236) and subcloned into vectors
pCLS15795 (SEQ ID NO: 351) and pCLS9013 (SEQ ID NO: 153) by
restriction and ligation using NcoI and NsiI restriction sites,
yielding TevD02_cT11Avr_FokI-L (pCLS15796, SEQ ID NO: 352, encoding
the protein of SEQ ID NO: 447) and TevD02_cT11Avr_TevD02
(pCLS15797, SEQ ID NO: 353, encoding the protein of SEQ ID NO:
448), respectively. All constructs were sequenced and the insert
transferred to additional vectors as needed (see below).
[0393] The final TevI::TALE-AvrBs3::FokI and
TevI::TALE-AvrBs3::TevI yeast expression plasmids, pCLS13299 (SEQ
ID NO: 354, encoding the protein of SEQ ID NO: 449) and pCLS13301
(SEQ ID NO: 355, encoding the protein of SEQ ID NO: 450), were
prepared by yeast in vivo cloning using plasmids pCLS15796 (SEQ ID
NO: 352) and pCLS15797 (SEQ ID NO: 353), respectively. To generate
an intact coding sequence by in vivo homologous recombination,
approximately 40 ng of each plasmid linearized by digestion with
BssHII and 1 ng of the pCLS0542 (SEQ ID NO: 156) plasmid DNA
linearized by digestion with NcoI and EagI were used to transform,
respectively, the yeast S. cerevisiae strain FYC2-6A (MATa,
trp1.DELTA.63, leu2.DELTA.1, his3.DELTA.200) using a high
efficiency LiAc transformation protocol (Arnould et al. 2007).
[0394] All the yeast target reporter plasmids containing the TALEN
DNA target sequences were constructed as previously described
(International PCT Applications WO 2004/067736 and in Epinat,
Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et
al. 2006; Smith, Grizot et al. 2006).
[0395] The TevI::TALE-AvrBs3::FokI and TevI::TALE-AvrBs3::TevI
constructs were tested in a yeast SSA assay as previously described
(International PCT Applications WO 2004/067736 and in Epinat,
Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et
al. 2006; Smith, Grizot et al. 2006) on pseudo palindromic targets
in order to compare activity with a standard TALE-AvrBs3::FokI
TALEN (pCLS8590, SEQ ID NO: 244), which requires two binding sites
for activity. AvrBs3 targets contain two identical recognition
sequences juxtaposed with the 3' ends proximal and separated by
"spacer" DNA ranging from 5 to 40 bps (SEQ ID NO: 157 to 192, Table
6). In addition, constructs were tested on a target having only a
single AvrBs3 or RagT2-R recognition site (SEQ ID NO: 238, Table
11). On suitable targets, the TevI::TALE-AvrBs3::FokI and
TevI::TALE-AvrBs3::TevI activity levels in yeast were comparable to
those of their parent molecules lacking the N-terminal
I-TevI-derived catalytic domain. Significant activity is
illustrated in table 11 for a sample single-site target, according
to the dcTALEN of the present invention.
TABLE-US-00010 TABLE 11 Activity of various cTALENs and dcTALENs on
dual- and single-site DNA targets. Target DNA Avr25RAGT2R Avr25
(dual-site) (single-site) TALEN Construct (SEQ ID NO: 177) (SEQ ID
NO: 238) TevI::TALE-AvrBs3 ++++ +++ TALE-AvrBs3::FokI ++++ n.d.
TALE-AvrBs3::TevI ++++ n.d. TevI::TALE-AvrBs3::FokI ++++ +++
TevI::TALE-AvrBs3::TevI ++++ +++ Relative activity is scaled as:
n.d., no activity detectable; +, <25% activity; ++, 25% to
<50% activity; +++, 50% to <75% activity; ++++, 75% to 100%
activity.
Example 4b
scTrex2::TALE::FokI Dual Cleavage TALEN
[0396] A dual cleavage TALEN(CD::TALE::CD), possessing an
N-terminal scTrex2-derived catalytic domain and a C-terminal
catalytic domain derived from Fokl, was generated on the baseline
bT2-Avr (SEQ ID NO: 137) scaffold. The catalytic domain fragment of
scTrex2 was excised from plasmid pCLS15798 (SEQ ID NO: 356,
encoding the protein of SEQ ID NO: 451) and subcloned into vector
pCLS15795 (SEQ ID NO: 351) by restriction and ligation using NcoI
and NsiI restriction sites, yielding scTrex2_cT11Avr_FokI-L
(pCLS15799, SEQ ID NO: 357, encoding the protein of SEQ ID NO:
452). The construct was sequenced and the insert transferred to
additional vectors as needed (see below).
[0397] DNA encoding the TALE-AvrBs3::FokI or
scTrex2::TALE-AvrBs3::FokI constructs from either pCLS15795 (SEQ ID
NO: 351) or pCLS15799 (SEQ ID NO: 357), respectively, was subcloned
into the pCLS1853 (SEQ ID NO: 193) mammalian expression plasmid
using Ascl and XhoI restriction enzymes for the receiving plasmid
and BssHII and XhoI restriction enzymes for the inserts, leading to
the mammalian expression plasmids pCLS14972 and pCLS14971 (SEQ ID
NO: 358 and 359), respectively.
[0398] All mammalian target reporter plasmids containing the TALEN
DNA target sequences were constructed using the standard Gateway
protocol (INVITROGEN) into a CHO reporter vector (Arnould, Chames
et al. 2006, Grizot, Epinat et al. 2010). The TALE-AvrBs3::FokI and
scTrex2::TALE-AvrBs3::Fokl constructs were tested in an
extrachromosomal assay in mammalian cells (CHO K1) on pseudo
palindromic targets in order to compare activity with a standard
TALE-AvrBs3::FokI TALEN, which requires two binding sites for
activity. AvrBs3 targets contain two identical recognition
sequences juxtaposed with the 3' ends proximal and separated by
"spacer" DNA ranging from 5 to 40 bps (SEQ ID NO: 157 to 192, Table
6).
[0399] For this assay, CHO K1 cells were transfected in a 96-well
plate format with 75 ng of target vector and an increasing quantity
of each variant DNA from 0.7 to 25 ng, in the presence of PolyFect
reagent (1 .mu.L per well). The total amount of transfected DNA was
completed to 125 ng (target DNA, variant DNA, carrier DNA) using an
empty vector. Seventy-two hours after transfection, culture medium
was removed and 150 .mu.l of lysis/revelation buffer for
.beta.-galactosidase liquid assay was added. After incubation at
37.degree. C., optical density was measured at 420 nm. The entire
process is performed on an automated Velocityll BioCel platform
(Grizot, Epinat et al. 2009).
[0400] Activity levels in mammalian cells on suitable targets for
the scTrex2::TALE-AvrBs3::Fokl construct were comparable to those
of the parent TALE-AvrBs3::FokI molecule, indicating that the extra
scTrex2 moiety does not impair the TALEN DNA cleavage function.
Assessment of the scTrex2 function is performed in assays suitable
for the detection of NHEJ events.
Example 5
[0401] Baseline designs for the cTALEN scaffolds are based on
established TALE DNA binding domains. Compact TALENs are designed
to be as small and efficient as possible. To obtain this goal it
may therefore be necessary to enlist "enhancer" domains to bridge
the functional gap between compact TALE DNA binding domains and the
various catalytic domains. FIG. 6 (A-E) illustrates various
non-exhaustive configurations wherein such enhancer domains can be
applied. Note that the figure is illustrative only, and N-- vs.
C-terminal variations are implied (i.e. FIG. 6A can also have an
N-terminal enhancer domain and C-terminal catalytic domain). Tables
1 and 2 lists potential enhancer domains that could assist in DNA
binding (specific and non-specific contacts).
[0402] Enhanced TALENs (eTALENs) are created using functional
cTALENS from Example 3. The addition of the enhancer domain is
evaluated in our yeast assay (see Example 1). A particular enhancer
domain is judged useful if it provides a minimal 5% enhancement in
efficiency of the starting cTALEN, more preferably a minimal 10%
enhancement, more preferably 20%, more preferably 30%, more
preferably 40%, more preferably 50%, again more preferably an
enhancement greater than 50%.
Example 5a
TALE::ColE7::TALE Enhanced TALENs
[0403] Enhanced TALENs (TALE::CD::TALE), possessing N- and
C-terminal TALE DNA binding domains bordering a central DNA
cleavage domain, were generated using the sT2 (SEQ ID NO: 135) core
scaffold. The layout of this class of compact TALEN is illustrated
in FIG. 6B, wherein the N-terminal "enhancer domain" is itself a
TALE DNA binding domain. A point-mutant derivative of the ColE7
catalytic domain (pCLS15785, SEQ ID NO: 285) was chosen for the
catalytic core of the eTALEN. Two final constructs,
TALE-AvrBs3::ColE7_A497::TALE-RagT2-R (pCLS15800, SEQ ID NO: 360,
encoding the protein of SEQ ID NO: 453) and
TALE-RagT2-R::ColE7_A497::TALE-AvrBs3 (pCLS15801, SEQ ID NO: 361,
encoding the protein of SEQ ID NO: 454), were obtained using
standard molecular cloning techniques with DNA sequences from sT2
(SEQ ID NO: 135), pCLS15785 (SEQ ID NO: 285), AvrBs3 (SEQ ID NO:
152) and RagT2-R (SEQ ID NO: 271) as templates. All TALE::CD::TALE
constructs were sequenced and the inserts transferred to additional
vectors as needed (see below).
[0404] The final TALE::CD::TALE-based yeast expression plasmids,
pCLS12106 (SEQ ID NO: 362) and pCLS12110 (SEQ ID NO: 363, were
prepared by restriction and ligation using NcoI and EagI
restriction sites to subclone into the pCLS0542 (SEQ ID NO: 156)
plasmid. The yeast S. cerevisiae strain FYC2-6A (MAT.alpha.,
trp1.DELTA.63, leu2.DELTA.1, his3.DELTA.200) was transformed using
a high efficiency LiAc transformation protocol (Arnould et al.
2007).
[0405] All the yeast target reporter plasmids containing the TALEN
DNA target sequences were constructed as previously described
(International PCT Applications WO 2004/067736 and in Epinat,
Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et
al. 2006; Smith, Grizot et al. 2006).
[0406] The TALE::CD::TALE constructs are tested in a yeast SSA
assay as previously described (International PCT Applications WO
2004/067736 and in Epinat, Arnould et al. 2003; Chames, Epinat et
al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al. 2006)
on asymmetric AvrBs3/RagT2-R hybrid targets in order to compare
activity with a parent compact TALEN (e.g. pCLS8589, SEQ ID NO:
233), which has activity on targets with a single binding site. In
addition, constructs are tested on a target having only a single
AvrBs3 or RagT2-R recognition site.
Example 6
[0407] To date, all known TAL effectors and derivatives thereof
appear to require a T base at positions -1 (FIG. 3) in the
recognition sequence. To overcome this limitation, an enhancer
domain is used to replace the N-terminal region of the TALE
protein. Sequence and structure-based homology modeling of the
N-terminal TALE region of bT2-derivatives have yielded three
potential candidate proteins (Table 1): (i) Fem-3 binding factor,
SEQ ID NO: 4, (FBF1, Puf family of RNA binding proteins) from C.
elegans; (ii) artificial alpha-helicoidal repeat proteins (aRep),
SEQ ID NO: 5 and; (iii) proteins of the Ankyrin super-family. The
content and arrangement of secondary structure elements allows for
using these models as starting points for enhancer domains that
replace the N-terminal region of the TALE protein.
[0408] Chimeric proteins are constructed using the analogous
regions from one of the 3 candidates mentioned to replace the
N-terminal TALE protein region up to the first canonical repeat
domain. The new interface is redesigned in silico, using the
homology models as guides. This approach can be used to pinpoint
the determinants of specificity for the requisite T at position -1
of the target sequence. The replacement enhancer domain should at
minimum provide structural integrity to the cTALEN protein.
Constructs are evaluated in our yeast assay (see Example 1). A
particular enhancer domain is judged useful if it provides a
minimal 5% retention in activity of the starting cTALEN in the
absence of a T at target position -1, more preferably a minimal 10%
retention, more preferably 20%, more preferably 30%, more
preferably 40%, more preferably 50%, again more preferably a
retention in activity greater than 50%.
Example 7
[0409] To generate more suitable and compact scaffolds for cTALENS,
the nature of the C-terminal region (beyond the final half-repeat
domain) of the TALE protein has been analyzed. Sequence and
structure-based homology modeling of the C-terminal TALE region of
bT2-derivatives have yielded three potential candidate proteins
(Table 1): (i) the hydrolase/transferase of Pseudomonas
Aeuriginosa, SEQ ID NO: 6; (ii) the Polymerase domain from the
Mycobacterium tuberculosis Ligase D, SEQ ID NO: 7; (iii) initiation
factor eIF2 from Pyrococcus, SEQ ID NO: 8; (iv) Translation
Initiation Factor Aif2betagamma, SEQ ID NO: 9. As in example 6,
homology models are used to pinpoint regions for generating
possible C-terminal truncations; potential truncation positions
include 28, 40, 64, 118, 136, 169, 190 residues remaining beyond
the last half-repeat domain. Additionally, homologous regions from
the aforementioned proteins can be used to replace the C-terminal
domain entirely. Contact prediction programs can be used to
identify, starting from the primary sequence of a protein, the
pairs of residues that are likely proximal in the 3D space. Such
chimeric proteins should provide more stable scaffolds on which to
build cTALENs.
[0410] Constructs are evaluated in our yeast assay (see Example 1).
A particular enhancer domain is judged useful if it provides a
minimal 5% retention in activity of the starting cTALEN, more
preferably a minimal 10% retention, more preferably 20%, more
preferably 30%, more preferably 40%, more preferably 50%, again
more preferably a retention in activity greater than 50%.
Example 8
[0411] To generate compact TALENS with alternative activities,
trans cTALENS are generated by (a) using a catalytic domain with
separable activities (FIG. 7A, B), or; (b) providing an auxiliary
activity as a TALE-fusion (FIG. 7C). Sequence and structure-based
modeling of class III (Chan, Stoddard et al. 2011). TypellS
restriction endonucleases (REases) were used to create trans TALENs
(see Table 2 for a non-exhaustive list). The initial trans TALEN is
generated via fusion of an independently active catalytic domain
(e.g. the Nt.BspD6I nickase) as described in Examples 3 and 4. In
principle this trans TALEN can be used as is depending on the
application. To convert the cTALEN to a functional trans TALEN, the
auxiliary domain (in this case, ss.BspD6I) is provided in trans
(FIG. 8A). Such optionally trans and/or heterodimeric proteins can
allow for cTALEN scaffolds with activity that can be modulated to a
given application.
[0412] Constructs are evaluated in our yeast assay (see Example 1).
A particular auxiliary domain is judged useful if it provides an
alternative activity to that of the starting cTALEN.
[0413] If the auxiliary domain used exhibits activity independent
of the initial cTALEN (i.e. in a non-trans TALEN context), it can
as well be fused to a TALE domain for specific targeting (FIG. 7B).
Auxiliary domains can also be provided in trans as targeted
entities to provide functions unrelated to the cTALEN (FIG.
7C).
Example 8a
Specific Inhibition of TALEN Catalytic Activity
[0414] As mentioned in examples 3c and 3d, both NucA (SEQ ID NO:
26) and ColE7 (SEQ ID NO: 140) can be inhibited by complex
formation with their respective inhibitor proteins, NuiA (SEQ ID
NO: 229) and Im7 (SEQ ID NO: 230). Colicin-E9 (SEQ ID NO: 366) is
another non-limiting example of protein which can be inhibited by
its respective inhibitor Im9 (SEQ ID NO: 369). With respect to
TALENs derived from the NucA (TALE::NucA) or ColE7 (TALE::ColE7)
catalytic domains, the inhibitors serve as auxiliary domains (FIG.
7A) that modulate the activity by preventing DNA cleavage.
[0415] The Im7 (SEQ ID NO: 230) and NuiA (SEQ ID NO: 229) inhibitor
proteins were subcloned into the pCLS7763 backbone (SEQ ID NO: 241)
by restriction and ligation using NcoI and EagI restriction sites,
yielding pCLS9922 (SEQ ID NO: 242) and pCLS9923 (SEQ ID NO: 243),
respectively. These plasmids were then used in co-transformation
experiments in the standard yeast SSA assay as previously described
(International PCT Applications WO 2004/067736 and in Epinat,
Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et
al. 2006; Smith, Grizot et al. 2006).
[0416] All the yeast target reporter plasmids containing the TALEN
DNA target sequences were constructed as previously described
(International PCT Applications WO 2004/067736 and in Epinat,
Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et
al. 2006; Smith, Grizot et al. 2006).
[0417] TALE-AvrBs3::NucA (pCLS9924, SEQ ID NO: 223) and
TALE-AvrBs3::ColE7 (pCLS8589, SEQ ID NO: 233) constructs were
tested in a yeast SSA assay on pseudo palindromic targets in order
to compare activity with a standard TALE-AvrBs3::FokI TALEN
(pCLS8590, SEQ ID NO: 244), which requires two binding sites for
activity. AvrBS3 targets contain two identical recognition
sequences juxtaposed with the 3' ends proximal and separated by
"spacer" DNA ranging from 5 to 40 bps (SEQ ID NO: 157 to 192, Table
7). In addition, constructs were tested on a target having only a
single AvrBs3 recognition site (SEQ ID NO: 224, Table 7). Activity
modulation of the TALENs was assessed in the presence or absence of
specific or unspecific inhibitor protein, using the
TALE-AvrBs3::FokI TALEN as control.
[0418] Data summarized in table 12 indicate that TALE-AvrBs3::NucA
and TALE-AvrBs3::ColE7 constructs are specifically inactivated by
the presence of their respective inhibitor proteins NuiA and Im7,
according to the present invention.
TABLE-US-00011 TABLE 12 Activity of TALEN constructs in the
presence of inhibitor protein. Inhibitor Protein TALEN Construct
None NuiA Im7 TALE-AvrBs3::NucA (SEQ ID NO: 223) ++++ n.d. ++++
TALE-AvrBs3::ColE7 (SEQ ID NO: 233) ++++ ++++ n.d.
TALE-AvrBs3::FokI (SEQ ID NO: 244) ++++ ++++ ++++ Relative activity
is scaled as: n.d., no activity detectable; +, <25% activity;
++, 25% to <50% activity; +++, 50% to <75% activity; ++++,
75% to 100% activity.
Example 8b
Enhancing TALEN Catalytic Activity Via a Trans TALEN
[0419] Example 3b illustrates that the TevI::TALE functions
unassisted as a compact TALEN (pCLS8522, SEQ ID NO: 237). To
further enhance activity, a trans TALEN was designed using a
TALE::TevI construct in a layout depicted in FIG. 7C. The DNA
sequence coding for the RVDs to target the RagT2-R site (SEQ ID NO:
271) was subcloned into plasmid pCLS7865-cT11_TevD02 (pCLS9011, SEQ
ID NO: 151) using Type IIS restriction enzymes BsmBI for the
receiving plasmid and BbvI and SfaNI for the inserted RVD sequence
to create the subsequent TALE-RagT2-R::TevI construct
cT11RagT2_R_TevD02 (pCLS15802, SEQ ID NO: 364). The construct was
sequenced and the insert subcloned into the pCLS7763 backbone (SEQ
ID NO: 241) by restriction and ligation using NcoI and EagI
restriction sites, yielding pCLS8990 (SEQ ID NO: 365). Plasmid
pairs pCLS8522 (SEQ ID NO: 237) and pCLS7763 (SEQ ID NO: 241) or
pCLS8522 (SEQ ID NO: 237) and pCLS8990 (SEQ ID NO: 365) were then
used in co-transformation experiments in the standard yeast SSA
assay as previously described (International PCT Applications WO
2004/067736 and in Epinat, Arnould et al. 2003; Chames, Epinat et
al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al.
2006).
[0420] All the yeast target reporter plasmids containing the TALEN
DNA target sequences were constructed as previously described
(International PCT Applications WO 2004/067736 and in Epinat,
Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et
al. 2006; Smith, Grizot et al. 2006).
[0421] The TALE-RagT2-R::TevI/TevI::TALE-AvrBs3 construct pairs
were tested in a yeast SSA assay as previously described
(International PCT Applications WO 2004/067736 and in Epinat,
Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et
al. 2006; Smith, Grizot et al. 2006) on asymmetric RagT2-R/AvrBs3
hybrid targets in order to compare activity with a parent compact
TALEN (e.g. pCLS8522, SEQ ID NO: 237), which has activity on
targets with a single binding site. RagT2-R/AvrBs3 hybrid targets
contain two different recognition sequences juxtaposed with the 3'
end of the first (RagT2-R) proximal to the 5' end of the second
(AvrBs3) and separated by "spacer" DNA ranging from 5 to 40 bps
(SEQ ID NO: G064 to G099, Table 13). FIG. 18 illustrates the
modulation in TevI::TALE-AvrBs3 activity provided by the
TALE-RagT2-R::TevI construct, according to the trans cTALEN of the
present invention.
Example 9
Replacement of the C-Terminal Domain by a Polypeptide Linker,
Activity with colE7 Catalytic Domain
[0422] We generated a first library of 37 different linkers. Many
of them have a common structure comprising a variable region
encoding 3 to 28 amino acids residues and flanked by regions
encoding SGGSGS stretch (SEQ ID NO: 219) at both the 5' and a 3'
end (SEQ ID NO: 372 to 408). These linkers contain XmaI and BamHI
restriction sites in their 5' and 3' ends respectively. The linker
library is then subcloned in pCLS7183 (SEQ ID NO: 141) via the XmaI
and BamHI restriction sites to replace the C-terminal domain of the
AvrBs3-derived TALEN (pCLS7184, SEQ ID NO: 196). The AvrBs3-derived
set of repeat domains (RVDs) or any other RVD sequences having or
lacking the terminal half RVD is cloned in this backbone library.
DNA from the library is obtained, after scrapping of the colonies
from the Petri dishes, using standard miniprep techniques. The FokI
catalytic head is removed using BamHI and EagI restriction enzymes,
the remaining backbone being purified using standard gel extraction
techniques. DNA coding for ColE7 catalytic domain (SEQ ID NO: 11)
was amplified by the PCR to introduce, at the DNA level, a BamHI
(at the 5' of the coding strand) and a EagI (at the 3' of the
coding strand) restriction site and, at the protein level, a linker
(for example -SGGSGS- stretch, SEQ ID NO: 219) between the C
terminal domain library and the catalytic head. After BamHI and
EagI digestion and purification, the DNA coding for the different
catalytic heads were individually subcloned into the library
scaffold previously prepared.
[0423] DNA from the final library is obtained, after scrapping of
the colonies from Petri dishes, using standard miniprep techniques
and the resulting libraries are screened in our yeast SSA assay as
previously described (International PCT Applications WO 2004/067736
and in Epinat, Arnould et al. 2003; Chames, Epinat et al. 2005;
Arnould, Chames et al. 2006; Smith, Grizot et al. 2006) on pseudo
palindromic targets in order to compare activity with a standard
TALE-AvrBs3::FokI TALEN, which requires two binding sites for
activity. AvrBs3 targets contain two identical recognition
sequences juxtaposed with the 3' ends proximal and separated by
"spacer" DNA containing 15, 18, 21 and 24 bps (SEQ ID NO: 167, 170,
173 and 176, Table 7). In addition, constructs (SEQ ID NO: 416-419)
were tested on a target having only a single AvrBs3 recognition
site (SEQ ID NO: 224). Data summarized in FIG. 20 show sequences of
the linker of a fraction of ColE7 constructs being active on
targets having two AvrBs3 recognition sites or only one AvrBs3
recognition site.
[0424] The U.S. provisional applications to which this application
claims priority as well as the corresponding PCT application being
filed Apr. 5, 2012 and entitled "METHOD FOR THE GENERATION OF
COMPACT TALE-NUCLEASES AND USES THEREOF" are hereby incorporated by
reference in their entireties.
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TABLE-US-00012 [0609] MEGA
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References